Compositions of fibroin peptides and protein fragments

Abstract

The present disclosure relates to peptide compositions, such as fibroin-derived peptide compositions.

Description

Technical Field

[0001] The present disclosure relates to peptide compositions, such as fibroin-derived peptide compositions. The present disclosure also relates to synthetic fabrics coated with fibroin and protein fragments. In further embodiments, the present disclosure relates to fibroin compositions and methods for stimulating collagen and / or connexin-1 expression. Background Art

[0002] Silk is a natural polymer produced by various insects and spiders and comprises a core protein, fibroin, and a gelatinous coating composed of a non-silk protein, sericin. Summary of the Invention

[0003] The present disclosure provides peptide compositions, such as fibroin-derived peptide compositions.

[0004] The present disclosure provides a kind of peptide or protein fragment, it comprises a plurality of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P and W, wherein at least one of amino acids is modified, substituted or replaced. In some embodiments, the peptide or protein fragment is a fibroin peptide or protein fragment comprising an amino acid modification, replacement or replacement of an amino acid selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, S, H, P and W. In some embodiments, fibroin is a fibroin heavy chain, a fibroin light chain or a fibroin hexamer (fibrohexamerin). In some embodiments, the peptide or protein fragment comprises about 2 to about 100 amino acids. In some embodiments, the peptide or protein fragment comprises about 2 to about 25 amino acids. In some embodiments, the peptide or protein fragment comprises about 25 to about 50 amino acids. In some embodiments, the peptide or protein fragment comprises about 50 to about 75 amino acids. In some embodiments, the peptide or protein fragment comprises about 75 to about 100 amino acids. In some embodiments, the peptide or protein fragment comprises about 100 to about 125 amino acids. In some embodiments, the peptide or protein fragment comprises about 125 to about 150 amino acids. In some embodiments, the peptide or protein fragment comprises about 150 to about 200 amino acids. In some embodiments, the peptide or protein fragment comprises about 200 to about 250, 300, 350, 400, 450 or 500 amino acids. In some embodiments, the peptide or protein fragment comprises one to five modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises one modification, substitution and / or replacement. In some embodiments, the peptide or protein fragment comprises two modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises three modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises four modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises five modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises six modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises seven modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises eight modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises nine modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises ten modifications, substitutions and / or replacements. In some embodiments, the modifications, substitutions and / or replacements are selected from asparagine to aspartic acid modifications, substitutions and / or replacements, glutamine to glutamic acid modifications, substitutions and / or replacements, and methionine to methionine oxide modifications, substitutions and / or replacements.In some embodiments, the fibroin is a fibroin heavy chain, and the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the fibroin heavy chain. In some embodiments, the modification, substitution and / or replacement is at Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262 of the fibroin heavy chain. In some embodiments, the fibroin is a fibroin light chain, and the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain. In some embodiments, the modification, substitution and / or replacement is located at N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255 of the silk fibroin light chain. In some embodiments, the silk fibroin is a silk fibroin hexamer (p25), and the modification, substitution and / or replacement is located at a position corresponding to any one of positions 1 to 220 of the silk fibroin hexamer (p25). In some embodiments, the modification, substitution and / or replacement is located at Q62, N93, M120, N149, N172, N174 and / or N202 of the silk fibroin hexamer (p25).

[0005] The present disclosure provides a composition comprising a plurality of peptides or protein fragments, each peptide or protein fragment comprising a plurality of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, S, H, P and W, wherein at least one of the amino acids is modified, substituted or replaced. In some embodiments, a plurality of peptides or protein fragments comprise amino acid modifications, substitutions or replacements of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, S, H, P and W. In some embodiments, the fibroin is a fibroin heavy chain, a fibroin light chain or a fibroin hexamer. In some embodiments, the peptide or protein fragment comprises about 2 to about 100 amino acids. In some embodiments, the peptide or protein fragment comprises about 2 to about 25 amino acids. In some embodiments, the peptide or protein fragment comprises about 25 to about 50 amino acids. In some embodiments, the peptide or protein fragment comprises about 50 to about 75 amino acids. In some embodiments, the peptide or protein fragment comprises about 75 to about 100 amino acids. In some embodiments, the peptide or protein fragment comprises about 100 to about 125 amino acids. In some embodiments, the peptide or protein fragment comprises about 125 to about 150 amino acids. In some embodiments, the peptide or protein fragment comprises about 150 to about 200 amino acids. In some embodiments, the peptide or protein fragment comprises about 200 to about 250, 300, 350, 400, 450 or 500 amino acids. In some embodiments, the peptide or protein fragment comprises one to five modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises one modification, substitution and / or replacement. In some embodiments, the peptide or protein fragment comprises two modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises three modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises four modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises five modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises six modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises seven modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises eight modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises nine modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises ten modifications, substitutions and / or replacements. In some embodiments, the modifications, substitutions and / or replacements are selected from asparagine to aspartic acid modifications, substitutions and / or replacements, glutamine to glutamic acid modifications, substitutions and / or replacements, and methionine to methionine oxide modifications, substitutions and / or replacements.In some embodiments, the fibroin is a fibroin heavy chain, and the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the fibroin heavy chain. In some embodiments, the modification, substitution and / or replacement is at Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262 of the fibroin heavy chain. In some embodiments, the fibroin is a fibroin light chain, and the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain. In some embodiments, the modification, substitution and / or replacement is located at N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255 of the fibroin light chain. In some embodiments, the fibroin is a fibroin hexamer (p25), and the modification, substitution and / or replacement is located at a position corresponding to any one of positions 1 to 220 of the fibroin hexamer (p25). In some embodiments, the modification, substitution and / or replacement is located at Q62, N93, M120, N149, N172, N174 and / or N202 of the fibroin hexamer (p25). In some embodiments, each modification, substitution and / or replacement is independently in the range of about 1% to about 99% in the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 1% to about 10% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 10% to about 20% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 20% to about 30% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 30% to about 40% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 40% to about 50% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 50% to about 60% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 60% to about 70% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 70% to about 80% of the composition. In some embodiments, each modification, substitution and / or replacement is independently in the range of about 80% to about 90% in the composition. In some embodiments, each modification, substitution and / or replacement is independently in the range of about 90% to about 99% in the composition.As used herein, % modification, % substitution and / or % replacement is defined as (the number of peptides or protein fragments comprising a modification, substitution and / or replacement at a particular position divided by the total number of peptides or protein fragments comprising the particular position, whether or not comprising a modification, substitution and / or replacement) x 100.

[0006] The present disclosure provides a composition comprising a plurality of peptides or protein fragments of a fibroin heavy chain, a fibroin light chain and / or a fibroin hexamer (p25), the composition comprising one or more fractions, wherein the plurality of peptides or protein fragments comprise fibroin peptides or protein fragments containing amino acid modifications, substitutions or replacements. In some embodiments, the plurality of peptides or protein fragments have a weight average molecular weight (MW) selected from about 1 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa or about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa or about 200 kDa to about 250 kDa. w ) and a polydispersity of 1 to about 1.7, about 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, or about 160 kDa to about 180 kDa. w ) and a polydispersity of 1 to about 1.1, or 1 to about 1.2, or 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ) and a polydispersity of 1 to about 1.1, or 1 to about 1.2, or 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 120 kDa. w) and a polydispersity of 1 to about 1.1, or 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ) and a polydispersity of 1 to about 1.1, or 1 to about 1.2, or 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ) and a polydispersity of 1 to about 1.1, or 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 20 kDa to about 40 kDa or about 40 kDa to about 60 kDa. w ) and a polydispersity of 1 to about 1.1, or 1 to about 1.2, or 1 to less than 3, or 1 to less than 5. In some embodiments, the one or more fractions are selected from AS77, AS78, AS79, AS80, and AS81. In some embodiments, the one or more fractions are selected from AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89. In some embodiments, the one or more fractions are selected from AS90, AS91, AS92, AS93, and AS94. In some embodiments, the one or more fractions are selected from AS95, AS96, AS97, AS98, AS99, and AS100. In some embodiments, the plurality of peptide or protein fragments have a weight average molecular weight (MW) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 220 kDa. w) and a polydispersity of 1 to about 1.7, or 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w ) and a polydispersity of 1 to about 1.2, or 1 to about 1.3, or 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ) and a polydispersity of 1 to about 1.1, or 1 to about 1.2, or 1 to less than 3, or 1 to less than 5. In some embodiments, the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w) and a polydispersity of 1 to about 1.2, or 1 to about 1.3, or 1 to less than 3, or 1 to less than 5. In some embodiments, one or more fractions are selected from AS101, AS102, AS103, AS104, and AS105. In some embodiments, one or more fractions are selected from AS106, AS107, AS108, AS109, AS110, and AS111. In some embodiments, the amino acids are selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W. In some embodiments, the peptide or protein fragment comprises from about 2 to about 100 amino acids. In some embodiments, the peptide or protein fragment comprises from about 2 to about 25 amino acids. In some embodiments, the peptide or protein fragment comprises from about 25 to about 50 amino acids. In some embodiments, the peptide or protein fragment comprises from about 50 to about 75 amino acids. In some embodiments, the peptide or protein fragment comprises about 75 to about 100 amino acids. In some embodiments, the peptide or protein fragment comprises about 100 to about 125 amino acids. In some embodiments, the peptide or protein fragment comprises about 125 to about 150 amino acids. In some embodiments, the peptide or protein fragment comprises about 150 to about 200 amino acids. In some embodiments, the peptide or protein fragment comprises about 200 to about 250, 300, 350, 400, 450, or 500 amino acids. In some embodiments, the peptide or protein fragment comprises one to five modifications, substitutions, and / or replacements. In some embodiments, the peptide or protein fragment comprises one modification, substitution, and / or replacement. In some embodiments, the peptide or protein fragment comprises two modifications, substitutions, and / or replacements. In some embodiments, the peptide or protein fragment comprises three modifications, substitutions, and / or replacements. In some embodiments, the peptide or protein fragment comprises four modifications, substitutions, and / or replacements. In some embodiments, the peptide or protein fragment comprises five modifications, substitutions, and / or replacements. In some embodiments, the peptide or protein fragment comprises six modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises seven modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises eight modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises nine modifications, substitutions and / or replacements. In some embodiments, the peptide or protein fragment comprises ten modifications, substitutions and / or replacements. In some embodiments, fibroin is a fibroin heavy chain, and wherein the modification, substitution and / or replacement is located at a position corresponding to any position in 1 to 5263 of the fibroin heavy chain. In some embodiments, fibroin is a fibroin light chain, and wherein the modification, substitution and / or replacement is located at a position corresponding to any position in 1 to 262 of the fibroin light chain.In some embodiments, the fibroin is a fibroin hexamer (p25) chain, and the modification, substitution and / or replacement is located at a position corresponding to any one of positions 1 to 220 of the fibroin hexamer (p25) chain. In some embodiments, the modification, substitution and / or replacement is selected from asparagine to aspartic acid modification, substitution and / or replacement, glutamine to glutamic acid modification, substitution and / or replacement, and methionine to methionine oxide modification, substitution and / or replacement. In some embodiments, the modification, substitution and / or replacement is located at a fibroin heavy chain position selected from Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262. In some embodiments, modification, substitution and / or replacement are located at a fibroin light chain position selected from N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255. In some embodiments, modification, substitution and / or replacement are located at a fibroin hexamer (p25) position selected from Q62, N93, M120, N149, N172, N174 and / or N202. In some embodiments, each modification, substitution and / or replacement is independently in the range of about 1% to about 99% in the composition. In some embodiments, each modification, substitution and / or replacement is independently in the range of about 1% to about 10% in the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 10% to about 20% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 20% to about 30% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 30% to about 40% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 40% to about 50% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 50% to about 60% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 60% to about 70% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 70% to about 80% of the composition. In some embodiments, each modification, substitution, and / or replacement is independently within the range of about 80% to about 90% of the composition. In some embodiments, each modification, substitution, and / or replacement independently ranges from about 90% to about 99% in the composition.As used herein, % modification, % substitution and / or % replacement is defined as (the number of peptides or protein fragments comprising a modification, substitution and / or replacement at a particular position divided by the total number of peptides or protein fragments that include the particular position, whether or not comprising a modification, substitution and / or replacement) x 100. In some embodiments, the molecular weight is determined by MALS.

[0007] The present disclosure provides a kind of article, it comprises one or more peptides or protein fragments disclosed herein and / or one or more compositions disclosed herein.Composition can comprise unmodified silk fibroin peptide and protein fragment.Article is selected from personal care products disclosed herein, coated fabric disclosed herein and / or article for treating fabric disclosed herein (for example, laundry beads).The present disclosure provides a kind of article, it comprises one or more peptides or protein fragments disclosed herein, and / or one or more compositions disclosed herein, and substrate disclosed herein or any other support or combination element.

[0008] In some embodiments, the present disclosure provides a composition disclosed herein comprising a plurality of peptides or protein fragments, e.g., a plurality of fibroin heavy chain peptides or fibroin heavy chain fragments, and comprising a plurality of amino acid modifications, substitutions and / or replacements, and further comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the group consisting of:

[0009] - a ratio of Q58:M64 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0010] - a ratio of Q58:N68 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0011] - a ratio of Q58:N70 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0012] - a ratio of Q58:N77 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0013] - a ratio of Q58:M80 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0014] - a ratio of Q58:N93 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0015] - a ratio of Q58:M103 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0016] - a ratio of Q58:Q125 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0017] - a ratio of Q58:N132 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0018] - a ratio of Q58:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0019] - a ratio of Q58:Q275 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0020] - a ratio of Q58:N4191 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0021] - a ratio of Q58:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0022] - a ratio of Q58:N5262 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0023] - a ratio of M64:N68 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0024] - a ratio of M64:N70 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0025] - a ratio of M64:N77 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0026] - a ratio of M64:M80 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0027] - a ratio of M64:N93 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0028] - a ratio of M64:M103 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0029] - a ratio of M64:Q125 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0030] - a ratio of M64:N132 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0031] - a ratio of M64:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0032] - a ratio of M64:Q275 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0033] - a ratio of M64:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0034] - a ratio of M64:Q5216 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0035] - a ratio of M64:N5262 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0036] - a ratio of N68:N70 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0037] - a ratio of N68:N77 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0038] - a ratio of N68:M80 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0039] - a ratio of N68:N93 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0040] - a ratio of N68:M103 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0041] - a ratio of N68:Q125 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0042] - a ratio of N68:N132 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0043] - a ratio of N68:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0044] - a ratio of N68:Q275 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0045] - a ratio of N68:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0046] - a ratio of N68:Q5216 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0047] - a ratio of N68:N5262 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0048] - a ratio of N70:N77 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0049] - a ratio of N70:M80 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0050] - a ratio of N70:N93 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0051] - a ratio of N70:M103 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0052] - a ratio of N70:Q125 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0053] - a ratio of N70:N132 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0054] - a ratio of N70:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0055] - a ratio of N70:Q275 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0056] - a ratio of N70:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0057] - a ratio of N70:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0058] - a ratio of N70:N5262 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0059] - a ratio of N77:M80 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0060] - a ratio of N77:N93 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0061] - a ratio of N77:M103 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0062] - a ratio of N77:Q125 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0063] - a ratio of N77:N132 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0064] - a ratio of N77:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0065] - a ratio of N77:Q275 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0066] - a ratio of N77:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0067] - a ratio of N77:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0068] - a ratio of N77:N5262 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0069] - a ratio of M80:N93 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0070] - a ratio of M80:M103 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0071] - a ratio of M80:Q125 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0072] - a ratio of M80:N132 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0073] - a ratio of M80:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0074] - a ratio of M80:Q275 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0075] - a ratio of M80:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0076] - a ratio of M80:Q5216 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0077] - a ratio of M80:N5262 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0078] - a ratio of N93:M103 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0079] - a ratio of N93:Q125 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0080] - a ratio of N93:N132 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0081] - a ratio of N93:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0082] - a ratio of N93:Q275 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0083] - a ratio of N93:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0084] - a ratio of N93:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0085] - a ratio of N93:N5262 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0086] - a ratio of M103:Q125 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0087] - a ratio of M103:N132 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0088] - a ratio of M103:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0089] - a ratio of M103:Q275 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0090] - a ratio of M103:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0091] - a ratio of M103:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0092] - a ratio of M103:N5262 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0093] - a ratio of Q125:N132 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0094] - a ratio of Q125:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0095] - a ratio of Q125:Q275 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0096] - a ratio of Q125:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0097] - a ratio of Q125:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0098] - a ratio of Q125:N5262 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0099] - a ratio of N132:Q139 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0100] - a ratio of N132:Q275 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0101] - a ratio of N132:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0102] - a ratio of N132:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0103] - a ratio of N132:N5262 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0104] - a ratio of Q139:Q275 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0105] - a ratio of Q139:N4191 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0106] - a ratio of Q139:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0107] - a ratio of Q139:N5262 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0108] - a ratio of Q275:N4191 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0109] - a ratio of Q275:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0110] - a ratio of Q275:N5262 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0111] - a ratio of N4191:Q5216 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0112] - a ratio of N4191:N5262 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0113] - a ratio of Q5216:N5262 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25.

[0114] In some embodiments, the present disclosure provides a composition comprising a plurality of peptides or protein fragments, such as a plurality of fibroin heavy and light chain peptides or fibroin heavy and light chain fragments, and comprising a plurality of amino acid modifications, substitutions and / or replacements, and further comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the following:

[0115] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain Q58 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0116] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain M64 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0117] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain N68 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0118] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain N70 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0119] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain N77 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0120] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain M80 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0121] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain N93 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0122] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain M103 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0123] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain Q125 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0124] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain N132 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0125] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain Q139 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0126] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain Q275 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0127] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain N4191 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0128] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of heavy chain Q5216 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements;

[0129] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 heavy chain N5262 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements.

[0130] In some embodiments, the present disclosure provides a composition comprising a plurality of peptides or protein fragments, for example, a plurality of fibroin heavy chains and p25 peptides or fibroin heavy chains and p25 fragments, and comprising a plurality of amino acid modifications, substitutions and / or replacements, comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the group consisting of:

[0131] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 heavy chain Q58 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0132] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20 or about 1:25 heavy chain M64 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0133] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 heavy chain N68 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0134] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20 or about 1:25 of heavy chain N70 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0135] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 heavy chain N77 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0136] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20 or about 1:25 heavy chain M80 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0137] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20, or about 1:25 heavy chain N93 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0138] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 heavy chain M103 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0139] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 heavy chain Q125 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0140] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 heavy chain N132 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0141] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 heavy chain Q139 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0142] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 heavy chain Q275 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0143] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20 or about 1:25 of heavy chain N4191 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0144] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 heavy chain Q5216 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0145] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20 or about 1:25 heavy chain N5262 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements.

[0146] In some embodiments, the present disclosure provides a composition comprising a plurality of peptides or protein fragments, for example, a plurality of light chain fibroin peptides or fibroin fragments, and comprising a plurality of amino acid modifications, substitutions and / or replacements, comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the following:

[0147] - a ratio of N23:Q24 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0148] - a ratio of Q24:N28 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0149] - a ratio of Q24:M69 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0150] - a ratio of Q24:N105 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0151] - a ratio of Q24:N108 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0152] - a ratio of Q24:N118 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0153] - a ratio of Q24:N136 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0154] - a ratio of Q24:N138 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0155] - a ratio of Q24:Q149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0156] - a ratio of Q24:N186 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0157] - a ratio of Q24:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0158] - a ratio of Q24:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0159] - a ratio of Q24:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0160] - a ratio of Q24:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0161] - a ratio of Q24:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0162] - a ratio of Q24:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0163] - a ratio of N28:M69 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0164] - a ratio of N28:N105 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0165] - a ratio of N28:N108 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0166] - a ratio of N28:N118 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0167] - a ratio of N28:N136 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0168] - a ratio of N28:N138 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0169] - a ratio of N28:Q149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0170] - a ratio of N28:N186 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0171] - a ratio of N28:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0172] - a ratio of N28:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0173] - a ratio of N28:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0174] - a ratio of N28:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0175] - a ratio of N28:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0176] - a ratio of N28:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0177] - a ratio of M69:N105 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0178] - a ratio of M69:N108 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0179] - a ratio of M69:N118 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0180] - a ratio of M69:N136 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0181] - a ratio of M69:N138 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0182] - a ratio of M69:Q149 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0183] - a ratio of M69:N186 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0184] - a ratio of M69:N200 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0185] - a ratio of M69:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0186] - a ratio of M69:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0187] - a ratio of M69:N240 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0188] - a ratio of M69:N248 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0189] - a ratio of M69:Q255 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0190] - a ratio of N105:N108 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0191] - a ratio of N105:N118 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0192] - a ratio of N105:N136 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0193] - a ratio of N105:N138 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0194] - a ratio of N105:Q149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0195] - a ratio of N105:N186 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0196] - a ratio of N105:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0197] - a ratio of N105:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0198] - a ratio of N105:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0199] - a ratio of N105:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0200] - a ratio of N105:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0201] - a ratio of N105:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0202] - a ratio of N108:N118 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0203] - a ratio of N108:N136 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0204] - a ratio of N108:N138 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0205] - a ratio of N108:Q149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0206] - a ratio of N108:N186 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0207] - a ratio of N108:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0208] - a ratio of N108:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0209] - a ratio of N108:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0210] - a ratio of N108:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0211] - a ratio of N108:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0212] - a ratio of N108:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0213] - a ratio of N118:N136 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0214] - a ratio of N118:N138 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0215] - a ratio of N118:Q149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0216] - a ratio of N118:N186 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0217] - a ratio of N118:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0218] - a ratio of N118:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0219] - a ratio of N118:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0220] - a ratio of N118:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0221] - a ratio of N118:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0222] - a ratio of N118:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0223] - a ratio of N136:N138 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0224] - a ratio of N136:Q149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0225] - a ratio of N136:N186 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0226] - a ratio of N136:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0227] - a ratio of N136:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0228] - a ratio of N136:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0229] - a ratio of N136:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0230] - a ratio of N136:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0231] - a ratio of N136:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0232] - a ratio of N138:Q149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0233] - a ratio of N138:N186 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0234] - a ratio of N138:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0235] - a ratio of N138:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0236] - a ratio of N138:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0237] - a ratio of N138:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0238] - a ratio of N138:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0239] - a ratio of N138:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0240] - a ratio of Q149:N186 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0241] - a ratio of Q149:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0242] - a ratio of Q149:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0243] - a ratio of Q149:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0244] - a ratio of Q149:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0245] - a ratio of Q149:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0246] - a ratio of Q149:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0247] - a ratio of N186:N200 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0248] - a ratio of N186:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0249] - a ratio of N186:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0250] - a ratio of N186:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0251] - a ratio of N186:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0252] - a ratio of N186:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0253] - a ratio of N200:Q202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0254] - a ratio of N200:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0255] - a ratio of N200:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0256] - a ratio of N200:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0257] - a ratio of N200:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0258] - a ratio of Q202:N204 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0259] - a ratio of Q202:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0260] - a ratio of Q202:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0261] - a ratio of Q202:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0262] - a ratio of N204:N240 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0263] - a ratio of N204:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0264] - a ratio of N204:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0265] - a ratio of N240:N248 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0266] - a ratio of N240:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0267] - a ratio of N248:Q255 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25.

[0268] In some embodiments, the present disclosure provides a composition comprising a plurality of peptides or protein fragments, such as a plurality of fibroin light and heavy chain peptides or fibroin light and heavy chain fragments, and comprising a plurality of amino acid modifications, substitutions and / or replacements, and further comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the group consisting of:

[0269] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain N23 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0270] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain Q24 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0271] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N28 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, or N5262 modifications, substitutions, and / or replacements;

[0272] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain M69 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0273] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain N105 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0274] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain N108 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0275] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N118 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, or N5262 modifications, substitutions, and / or replacements;

[0276] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain N136 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0277] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain N138 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0278] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain Q149 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0279] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain N186 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0280] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N200 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, or N5262 modifications, substitutions, and / or replacements;

[0281] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain Q202 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0282] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N204 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, or N5262 modifications, substitutions, and / or replacements;

[0283] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N240 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, or N5262 modifications, substitutions, and / or replacements;

[0284] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain N248 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0285] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of light chain Q255 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements.

[0286] In some embodiments, the present disclosure provides a composition comprising a plurality of peptides or protein fragments, for example, a plurality of fibroin light chains and p25 peptides or a fibroin heavy chain and a p25 fragment, and comprising a plurality of amino acid modifications, substitutions and / or replacements, comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the group consisting of:

[0287] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N23 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0288] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 light chain Q24 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0289] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N28 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0290] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20 or about 1:25 of light chain M69 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements;

[0291] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N105 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0292] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N108 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0293] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N118 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0294] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N136 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0295] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N138 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0296] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 light chain Q149 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0297] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N186 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0298] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20, or about 1:25 of light chain N200 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0299] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain Q202 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0300] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N204 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0301] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N240 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0302] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of light chain N248 to p25 Q62, N93, M120, N149, N172, N174, or N202 modifications, substitutions, and / or replacements;

[0303] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 light chain Q255 to p25 Q62, N93, M120, N149, N172, N174 or N202 modifications, substitutions and / or replacements.

[0304] In some embodiments, the present disclosure provides a composition comprising a plurality of peptides or protein fragments, for example, a plurality of p25 fibroin peptides or fibroin fragments, and comprising a plurality of amino acid modifications, substitutions and / or replacements, comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the group consisting of:

[0305] - a ratio of Q62:N93 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0306] - a ratio of Q62:M120 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0307] - a ratio of Q62:N149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0308] - a ratio of Q62:N172 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0309] - a ratio of Q62:N174 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0310] - a ratio of Q62:N202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0311] - a ratio of N93:M120 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0312] - a ratio of N93:N149 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0313] - a ratio of N93:N172 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0314] - a ratio of N93:N174 modifications, substitutions, and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25;

[0315] - a ratio of N93:N202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0316] - a ratio of M120:N149 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0317] - a ratio of M120:N172 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0318] - a ratio of M120:N174 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0319] - a ratio of M120:N202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0320] - a ratio of N149:N172 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0321] - a ratio of N149:N174 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0322] - a ratio of N149:N202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0323] - a ratio of N172:N174 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0324] - a ratio of N172:N202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25;

[0325] - a ratio of N174:N202 modifications, substitutions and / or replacements of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25.

[0326] In some embodiments, the present disclosure provides a composition comprising a plurality of peptides or protein fragments, e.g., a plurality of p25 and heavy chain peptides or p25 and heavy chain fragments, and comprising a plurality of amino acid modifications, substitutions and / or replacements, comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the group consisting of:

[0327] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of p25 Q62 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0328] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of p25 N93 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, or N5262 modifications, substitutions, and / or replacements;

[0329] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of p25 M120 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0330] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of p25 N149 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, or N5262 modifications, substitutions, and / or replacements;

[0331] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of p25 N172 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, or N5262 modifications, substitutions, and / or replacements;

[0332] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20 or about 1:25 of p25 N174 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements;

[0333] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20 or about 1:25 of p25 N202 to heavy chain Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 or N5262 modifications, substitutions and / or replacements.

[0334] In some embodiments, the present disclosure provides a composition comprising a plurality of peptides or protein fragments, such as a plurality of p25 and light chain peptides or fibroin light chains and light chain fragments, and comprising a plurality of amino acid modifications, substitutions and / or replacements, and further comprising modifications, substitutions and / or replacements at specific positions in one or more ratios selected from the following:

[0335] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of p25 Q62 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, or Q255 modifications, substitutions, and / or replacements;

[0336] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of p25 N93 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, or Q255 modifications, substitutions, and / or replacements;

[0337] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20, or about 1:25 of p25 M120 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, or Q255 modifications, substitutions, and / or replacements;

[0338] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of p25 N149 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, or Q255 modifications, substitutions, and / or replacements;

[0339] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of p25 N172 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, or Q255 modifications, substitutions, and / or replacements;

[0340] - a ratio of about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:25 of p25 N174 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, or Q255 modifications, substitutions, and / or replacements;

[0341] - a ratio of about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20 or about 1:25 of p25 N202 to light chain N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 or Q255 modifications, substitutions and / or replacements.

[0342] The present disclosure provides an article comprising a fabric and a coating, wherein the coating comprises a surfactant and / or emulsifier system, and a fibroin fragment having a thickness selected from the group consisting of about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 39 kDa, about 30 ... In some embodiments, the coating comprises an average weight average molecular weight of from about 1 to about 30 kDa, from about 30 kDa to about 35 kDa, from about 35 kDa to about 40 kDa, from about 39 kDa to about 54 kDa, from about 39 kDa to about 80 kDa, from about 40 kDa to about 45 kDa, from about 45 kDa to about 50 kDa, from about 50 kDa to about 55 kDa, from about 55 kDa to about 60 kDa, from about 60 kDa to about 100 kDa, or from about 80 kDa to about 144 kDa, and a polydispersity ranging from 1 to about 5. In some embodiments, the coating comprises a fibroin peptide and / or fibroin fragment disclosed herein, such as, but not limited to, a fibroin peptide and / or protein fragment, wherein at least one of the amino acids is modified, substituted, or replaced as disclosed herein. In some embodiments, the polydispersity of the fibroin fragments is from 1 to about 1.5, from about 1.5 to about 2.0, from about 2.0 to about 2.5, from about 2.5 to about 3.0, from about 3.0 to about 3.5, from about 3.5 to about 4.0, from about 4.0 to about 4.5, or from about 4.5 to about 5.0. In some embodiments, the polydispersity of the fibroin fragments is from about 1.5 to about 3.0. In some embodiments, the fibroin fragments include one or more of low molecular weight fibroin fragments and medium molecular weight fibroin fragments. In some embodiments, the article further comprises sericin in an amount of from about 0.01% (w / w) to about 10% (w / w) relative to the fibroin fragments. In some embodiments, the fabric comprises one or more of the following: polyester, polyamide, polyaramid, polytetrafluoroethylene, polyethylene, polypropylene, polyurethane, silicone, a mixture of polyurethane and polyethylene glycol, ultra-high molecular weight polyethylene, high performance polyethylene, nylon, LYCRA (polyester-polyurethane copolymer, also known as SPANDEX and elastomer), or mixtures thereof. In some embodiments, the coating further comprises one or more of a wetting agent, a defoaming agent, a softening agent, a wicking agent, and an antimicrobial agent.In some embodiments, the w / w ratio of the silk fibroin fragments to the surfactant and / or emulsifier system in the coating is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 79:22, about 79:23, about 79:24, about 79:25, about 79:26, about 79:27, about 79:28, about 79:29, about 79:21 ...9, about 79:21, about 79:22, about 79:23, about 79:24, about 79:25, about 79:26, about 79:27, about 79:28 About 78:22, about 77:23, about 76:24, about 75:25, about 74:26, about 73:27, about 72:28, about 71:29, about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53: 47, about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69, about 30:70, about 29:71, about 28:72, about : 98, or about 1: 99. In some embodiments, the w / w ratio of the fibroin fragments to the surfactant and / or emulsifier in the coating is about 1: 1, about 1:2, about 1:4, about 1:8, about 1:16, or about 1:32.In some embodiments, the w / w ratio of the silk fibroin fragments in the coating to the surfactant and / or emulsifier system is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, about 1:25, about 1:26, about 1:27, about 1:28, about 1:29, about 1:30, about 1:31 or about 1:32. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, polyoxyethylene castor oil, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: polyoxyethylene (10-30) sorbitan monooleate, polyoxyethylene (10-30) sorbitan trioleate, polyoxyethylene (10-50) castor oil, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan trioleate, polyoxyethylene (29) castor oil, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan tristearate, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: sorbitan monofatty acid, sorbitan trifatty acid, castor oil, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: cocoyl glucoside, decyl glucoside, lauryl glucoside, sucrose cocoate, octyl / octyl glucoside, octyl / octyl glucoside, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system has an HLB of about 11 to about 13.50. In some embodiments, the surfactant and / or emulsifier system has an HLB of from about 11 to about 11.50, from about 11.50 to about 12, from about 12 to about 12.50, from about 12.50 to about 13, or from about 13 to about 13.50. In some embodiments, the article has improved moisture management compared to a similar article comprising a similar fabric but without the coating.In some embodiments, moisture management is assessed by a water absorption test, a vertical wicking test, or a drying rate test. In some embodiments, the article has improved drape compared to a similar article comprising a similar fabric but without the coating. In some embodiments, the article has improved smoothness compared to a similar article comprising a similar fabric but without the coating. In some embodiments, the article has improved hand compared to a similar article comprising a similar fabric but without the coating. In some embodiments, the article has a lower charge density at a given pH value compared to a similar article comprising a similar fabric but without the coating.

[0343] The present disclosure provides a method for preparing a fibroin-coated fabric, the method comprising: applying a solution comprising a surfactant and / or an emulsifier system to the fabric; applying a solution of fibroin fragments to the fabric; and drying the fabric. In some embodiments, the method comprises using modified silk fragments disclosed herein, such as, but not limited to, fibroin peptides and / or protein fragments, wherein at least one of the amino acids is modified, substituted, or replaced as disclosed herein. The present disclosure also provides a method for preparing a fibroin-coated fabric, the method comprising: applying a solution comprising a surfactant and / or an emulsifier system and fibroin fragments to the fabric; and drying the fabric. In some embodiments, the concentration of the fibroin fragments in the solution ranges from 0.01 g / L to about 100 g / L. In some embodiments, the concentration of the surfactant and / or emulsifier system in the solution ranges from 0.01 g / L to about 100 g / L. In some embodiments, the fibroin fragments have an average weight average molecular weight selected from about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, or about 80 kDa to about 144 kDa, and a polydispersity ranging from 1 to about 5. In some embodiments, the polydispersity of the fibroin fragments is from 1 to about 1.5, from about 1.5 to about 2.0, from about 2.0 to about 2.5, from about 2.5 to about 3.0, from about 3.0 to about 3.5, from about 3.5 to about 4.0, from about 4.0 to about 4.5, or from about 4.5 to about 5.0. In some embodiments, the polydispersity of the fibroin fragments is from about 1.5 to about 3.0. In some embodiments, the fibroin fragments include one or more of low molecular weight fibroin fragments and medium molecular weight fibroin fragments. In some embodiments, the solution further comprises about 0.01% (w / w) to about 10% (w / w) sericin relative to the fibroin fragments. In some embodiments, the fabric comprises one or more of: polyester, polyamide, polyaramid, polytetrafluoroethylene, polyethylene, polypropylene, polyurethane, silicone, a mixture of polyurethane and polyethylene glycol, ultra-high molecular weight polyethylene, high-performance polyethylene, nylon, LYCRA (polyester-polyurethane copolymer, also known as SPANDEX and elastomer), or mixtures thereof.In some embodiments, the solution further comprises one or more of a wetting agent, a defoaming agent, a softening agent, a wicking agent, and an antimicrobial agent. In some embodiments, the w / w ratio of the silk fibroin fragment to the surfactant and / or emulsifier system is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 78:23, about 79:24, about 78:25, about 78:26, about 78:27, about 78:28 :22, about 77:23, about 76:24, about 75:25, about 74:26, about 73:27, about 72:28, about 71:29, about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47 , about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69, about 30:70, about 29:71, about 28:72, about 2 : 7:73, about 26:74, about 25:75, about 24:76, about 23:77, about 22:78, about 21:79, about 20:80, about 19:81, about 18:82, about 17:83, about 16:84, about 15:85, about 14:86, about 13:87, about 12:88, about 11:89, about 10:90, about 9:91, about 8:92, about 7:93, about 6:94, about 5:95, about 4:96, about 3:97, about 2:98, or about 1:99. In some embodiments, the w / w ratio of the fibroin fragments to the surfactant and / or emulsifier system is about 1:1, about 1:2, about 1:4, about 1:8, about 1:16, or about 1:32.In some embodiments, the w / w ratio of the fibroin fragments to the surfactant and / or emulsifier system is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, about 1:25, about 1:26, about 1:27, about 1:28, about 1:29, about 1:30, about 1:31 or about 1:32. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, polyoxyethylene castor oil, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: polyoxyethylene (10-30) sorbitan monooleate, polyoxyethylene (10-30) sorbitan trioleate, polyoxyethylene (10-50) castor oil, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan trioleate, polyoxyethylene (29) castor oil, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan tristearate, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: sorbitan monofatty acid, sorbitan trifatty acid, castor oil, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system comprises one or more of the following: cocoyl glucoside, decyl glucoside, lauryl glucoside, sucrose cocoate, octyl / octyl glucoside, octyl / octyl glucoside, and any combination thereof. In some embodiments, the surfactant and / or emulsifier system has an HLB of about 11 to about 13.50. In some embodiments, the surfactant and / or emulsifier system has an HLB of about 11 to about 11.50, about 11.50 to about 12, about 12 to about 12.50, about 12.50 to about 13, or about 13 to about 13.50. In some embodiments, the drying comprises heating. In some embodiments, the pH of the solution is acidic.In some embodiments, the pH of the solution is from about 3.5 to about 4, from about 4 to about 4.5, from about 4.5 to about 5, from about 5 to about 5.5, or from about 5.5 to about 6.

[0344] The present disclosure also provides articles prepared by the methods described herein, including but not limited to by using modified silk fragments as described herein, such as but not limited to fibroin peptides and / or protein fragments, wherein at least one of the amino acids is modified, substituted or replaced as disclosed herein. In some embodiments, the articles have improved moisture management compared to similar articles comprising similar fabrics but not containing coatings. In some embodiments, moisture management is assessed by water absorption test, vertical wicking test or drying rate test. In some embodiments, the articles have improved drapability compared to similar articles comprising similar fabrics but not containing coatings. In some embodiments, the articles have improved smoothness compared to similar articles comprising similar fabrics but not containing coatings. In some embodiments, the articles have improved feel compared to similar articles comprising similar fabrics but not containing coatings. In some embodiments, the articles have lower charge density at a given pH value compared to similar articles comprising similar fabrics but not containing coatings.

[0345] In some embodiments, the present disclosure also provides personal care and / or cosmetic formulations and / or compositions comprising fibroin fragments having a size selected from the group consisting of about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 15 kDa to about 20 kDa, about 14 kDa to about 30 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa. a, an average weight average molecular weight of about 35kDa to about 40kDa, about 39kDa to about 54kDa, about 39kDa to about 80kDa, about 40kDa to about 45kDa, about 45kDa to about 50kDa, about 60kDa to about 100kDa and about 80kDa to about 144kDa and a polydispersity of 1 to about 5, but not limited thereto, may also comprise modified silk fragments disclosed herein, such as, but not limited to, fibroin peptides and / or protein fragments, wherein at least one of the amino acids is modified, substituted or replaced as disclosed herein. In some embodiments, the composition further comprises 0 to 500ppm of lithium bromide. In some embodiments, the composition further comprises 0 to 500ppm of sodium carbonate. In some embodiments, the polydispersity of the fibroin fragments is 1 to about 1.5. In some embodiments, the polydispersity of the fibroin fragments is about 1.5 to about 2.0. In some embodiments, the polydispersity of the fibroin fragments is about 1.5 to about 3.0. In some embodiments, the polydispersity of the fibroin fragments is from about 2.0 to about 2.5. In some embodiments, the polydispersity of the fibroin fragments is from about 2.5 to about 3.0. In some embodiments, the fibroin fragments are present in the composition in an amount of from about 0.001% to about 10.0% by weight relative to the total weight of the composition. In some embodiments, the composition further comprises from about 0.001% (w / w) to about 10% (w / w) sericin relative to the total weight of the composition. In some embodiments, the composition further comprises from about 0.001% (w / w) to about 10% (w / w) sericin relative to the fibroin fragments. In some embodiments, the fibroin fragments do not spontaneously or gradually gel and do not change significantly in color or turbidity when in aqueous solution for at least 10 days prior to being formulated into the composition. In some embodiments, the fibroin fragments are present in the composition in an amount of from about 0.01% to about 10.0% by weight relative to the total weight of the composition. In some embodiments, the fibroin fragments are present in the composition in an amount of about 0.01 wt % to about 1.0 wt % relative to the total weight of the composition.In some embodiments, the fibroin fragments are present in the composition in an amount of about 1.0 wt % to about 2.0 wt % relative to the total weight of the composition. In some embodiments, the fibroin fragments are present in the composition in an amount of about 2.0 wt % to about 3.0 wt % relative to the total weight of the composition. In some embodiments, the fibroin fragments are present in the composition in an amount of about 3.0 wt % to about 4.0 wt % relative to the total weight of the composition. In some embodiments, the fibroin fragments are present in the composition in an amount of about 4.0 wt % to about 5.0 wt % relative to the total weight of the composition. In some embodiments, the fibroin fragments are present in the composition in an amount of about 5.0 wt % to about 6.0 wt % relative to the total weight of the composition.

[0346] In some embodiments, the composition is formulated as a topical composition. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a dermatologically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of a suspension, emulsion, powder, solution, dispersion or elixir. In some embodiments, the pharmaceutically acceptable carrier comprises or is formulated as one or more of a gel, jelly, cream, lotion, foam, slurry, ointment, oil, paste, suppository, spray, semi-solid composition, solid composition, stick or mousse. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of sesame oil, corn oil, cottonseed oil or peanut oil. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of mannitol or glucose. In some embodiments, the pharmaceutically acceptable carrier comprises free and / or uncross-linked hyaluronic acid. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of an aliphatic oil, a fatty alcohol, a fatty acid, a glyceride, an acylglycerol, and a phospholipid. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of a monoglyceride, a diglyceride, or a triglyceride. In some embodiments, the pharmaceutically acceptable carrier comprises an aqueous phase. In some embodiments, the pharmaceutically acceptable carrier comprises an oil-in-water emulsion or a water-in-oil emulsion. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of a hydrocarbon oil, a fatty acid, a fatty oil, a fatty acid ester, or a cationic quaternary ammonium salt. BRIEF DESCRIPTION OF THE DRAWINGS

[0347] The presently disclosed embodiments will be further explained with reference to the accompanying drawings. The drawings shown are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the presently disclosed embodiments.

[0348] Figure 1 Ion exchange sorting scheme for separating populations comprising low-slip and moderate-slip silk / modified polypeptide compositions. Low-slip and moderate-slip silk / modified polypeptide compositions contain negatively charged, positively charged, or neutral silk / modified polypeptides. These populations were separated using Q anion exchange chromatography (A).

[0349] Figure 2 .Chromatogram of a low-slip silk / modified peptide composition loaded in a Q-Sepharose HP column (Cytiva). The effluent contains silk / modified peptides that cannot be captured in the column and are depleted of negatively charged amino acids. After loading the column with a low-slip or medium-slip silk / modified peptide composition and collecting the effluent, the column was washed until the UV-280 absorbance became less than 200 AU. The captured negatively charged silk / modified peptides were eluted with a high salt concentration (1 M NaCl) and constituted AS11 and AS22. Chromatography was performed in a buffer containing Tris, but the effluent and Q-eluate were finally dialyzed in water.

[0350] Figure 3 Analytical size exclusion chromatography of low-slip, medium-slip / modified silk compositions and their component AS compositions. Average molecular weight in kDa and polydispersity measurements are shown.

[0351] Figures 4A-4B Analytical size exclusion chromatography of low- and medium-slippery silk / modified peptide compositions and their components (see Table 1 for more details). Figure 4A , the molecular weights of the various new active silk compositions described in this study. Figure 4B , polydispersity (PDI) of the various novel active silk compositions described in this study. AS24 reconstructed the average molecular weight and polydispersity of the low-slippery / modified peptide composition and is composed of 50% AS12 and 50% AS22 (see Table 1 for details). AS6 reconstructed the average molecular weight and polydispersity of the medium-slippery / modified peptide composition and is composed of 50% AS1 and 50% AS11 (see Table 1 for details).

[0352] Figure 5 Isoelectric focusing electrophoresis of low-slippery silk / modified peptide compositions. Lanes 2 and 7: Low-slippery silk, different loading amounts. Lanes 3, 5, 8, and 10: AS12 silk, different preparations with different loading amounts. Lanes 4, 6, 9, and 11: AS22 silk, different preparations with different loading amounts.

[0353] Figures 6A-6BSelf-assembly reactions of low- and medium-slip silk / modified peptide compositions and their components (see Table 1 for further details). Both figures depict the kinetic parameters of gel formation during silk self-assembly. In panel A, the calculations of three self-assembly kinetic parameters, t0.5, Amax, and SARF, are shown. See the text for further details.

[0354] Figures 7A-7C Self-assembly kinetics of low- and medium-slippery silk / modified peptide compositions and their components (see Table 1 for more details). Figure 7A , the self-assembly rate factor shows how fast the self-assembly reaction proceeds once it is initiated and the self-assembling cores are organized. Figure 7B , the maximum gel yield shows the density of the silk gel after self-assembly is completed. Figure 7C , the time required for the self-assembly reaction to produce half the maximum amount of gel.

[0355] Figure 8 Low-slippery and medium-slippery silk / modified peptide compositions and their components (see Table 1 for more details). The self-assembly factor reflects the average propensity of silk to self-assemble and form a gel.

[0356] Although the above-mentioned drawings illustrate the presently disclosed embodiments, as noted in the discussion, other embodiments are also contemplated. The present disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art that fall within the scope and spirit of the principles of the presently disclosed embodiments.

[0357] Figure 9 is a graph of the weight average molecular weight average (i.e., mean molecular weight average or mean MW) of silk fibroin dissolved in 9.3 M LiBr as a function of time at 100°C-103°C (i.e., a temperature gradient of 3 degrees Celsius between 100°C and 103°C) as plotted using size exclusion chromatography with a refractive index detector (SEC-RI).

[0358] Figure 10 is a graph of the weight average molecular weight average (i.e., average molecular weight average or average MW) of silk fibroin dissolved in 9.3M LiBr at 122°C-125°C (i.e., a temperature gradient of 3 degrees Celsius between 122°C and 125°C) as a function of time, which was plotted using size exclusion chromatography with a refractive index detector (SEC-RI).

[0359] Figure 11 is a graph showing the percentage of amino acid modifications in silk.

[0360] Figures 12A-12C is a graph showing the percentage of amino acid modifications in low-sliding and medium-sliding silk. Figure 12A Heavy chain modifications are shown, Figure 12BLight chain modifications are shown, and Figure 12C A modified silk fibroin hexamer is shown. N is an asparagine converted to aspartic acid, and Q is a deamidated glutamine. N corresponds to an oxidized methionine. The number following each amino acid indicates its position along the amino acid chain from the corresponding protein.

[0361] Figures 13A-13B is a graph showing the percentage of amino acid modifications in produced and lyophilized low- and medium-smooth silk. Figure 13A Heavy chain modifications are shown, and Figure 13B Light chain modifications are shown. N is asparagine converted to aspartic acid, and Q is deamidated glutamine. N corresponds to oxidized methionine. The number following each amino acid indicates its position along the amino acid chain from the corresponding protein.

[0362] Figures 14A-14B is a graph showing the percentage of amino acid modifications in low-slip silk produced at Walpole and Medford using the slippage process with different process parameters and variable levels. Figure 14A Heavy chain modifications are shown, and Figure 14B Light chain modifications are shown. N is asparagine converted to aspartic acid, and Q is deamidated glutamine. N corresponds to oxidized methionine. The number following each amino acid indicates its position along the amino acid chain from the corresponding protein.

[0363] Figures 15A-15D This graph shows the percentage of amino acid modifications in low- and medium-strength silk produced using the slide and benchtop processes. N represents asparagine converted to aspartic acid, and Q represents deamidated glutamine. N corresponds to oxidized methionine. The number following each amino acid indicates its position along the amino acid chain from the corresponding protein.

[0364] Figure 16 is an explanation of the method used to calculate the percentage of modified amino acids at specific positions along each peptide sequence.

[0365] Figure 17 Anion exchange chromatography and size exclusion chromatography schemes for separating low-slippery silk / modified peptide compositions are shown. Low-slippery silk / modified peptide compositions consist of diverse peptide populations with a wide range of sizes and charges. Different populations of low-slippery silk / modified peptide compositions were isolated using Q-Sepharose anion exchange chromatography as the first step and HiLoad Superdex 200 size exclusion chromatography as the second purification step. The Q-Sepharose eluate was loaded onto a HiLoad Superdex 200 size exclusion chromatograph, which resulted in size sorting of negatively charged silk compositions / modified peptides.

[0366] Figure 18A and 18B is a chromatogram of anion exchange chromatography and subsequent size exclusion chromatography of the eluate (Q-eluate) of the low-slipid / modified polypeptide composition. Figure 18A : Anion exchange chromatography was performed using a Q-Sepharose column (Cytiva). Anion exchange chromatography separated the low-slip silk / modified peptide composition into an uncharged peptide population (flowthrough - light blue background) and eluted negatively charged silk composition (eluate - light pink background). The light yellow background indicates that the column was washed with 50 mM Tris pH = 8.0 before eluting the charged peptide population. Figure 18B The negatively charged eluate was loaded onto a Superdex 200 column and passed through a column containing 50 mM Tris, 200 mM CaCl2, pH = 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the low-slip silk / modified peptide compositions by size. The relative elution volumes of silk compositions AS77 and AS81 are indicated on the chromatogram.

[0367] Figure 19A and 19B Analytical size exclusion chromatograms of the low slip silk / modified silk composition and its component AS composition are shown. Figure 19A The average molecular weight in kDa of low-slip silk (LS) and AS77-AS81 is shown. Figure 19B Polydispersity (PDI) measurements are shown. Numerical data are presented in Table 7.

[0368] Figure 20 This is an SDS-polyacrylamide gel electrophoresis of low-slippery silk / modified polypeptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, with their respective silk compositions being: fraction 6 is AS77, fraction 7 is AS78, fraction 8 is AS79, fraction 9 is AS80, and fraction 10 is AS81.

[0369] Figure 21A and 21B Graph showing the self-assembly reaction of a low-sliding / modified peptide composition. A medium-sliding reaction was used as a positive control. Figure 21A The kinetic parameters of gel formation during silk self-assembly are shown. The self-assembly parameters of the medium-sliding silk are: Amax is 0.6780 (Abs), SARF is 8.676, T0.5 is 3.668 h, and FSAF is 3.08 (Abs / min). Figure 21B This is a snapshot of the same self-assembly assay at a later time point, 12 days after setting up the assay. None of the fractions tested self-assembled over time.

[0370] Figure 22A and 22BCharacterization of low-slip silk compositions by dynamic light scattering is shown. Low-slip silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the diameter particle size of each silk composition. Figure 22A . Shown are the intensity diameter particle size distributions measured for silk compositions AS77, AS78, AS79, AS80 and AS81. Figure 22B Correlogram functions for silk compositions AS77, AS78, AS79, AS80, AS81 are shown.

[0371] Figure 23 A size exclusion chromatogram showing the separation of a low-slippery / modified peptide composition. The low-slippery / modified peptide composition consists of a variety of peptide populations with a wide range of sizes. Different populations of low-slippery / modified peptide compositions were separated using HiLoad Superdex 200 size exclusion chromatography.

[0372] Figure 24 This is a chromatogram of a low-slippery / modified peptide composition loaded onto a Superdex 200 gel filtration column. The low-slippery / modified peptide composition was loaded onto a Superdex 200 column and passed through a column containing 50 mM Tris, 200 mM CaCl2, pH 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the low-slippery / modified peptide composition by size. The relative elution volumes of silk compositions AS82, AS86, and AS87 are indicated on the chromatogram.

[0373] Figure 25A and 25B Analytical size exclusion chromatograms of the low slip silk / modified silk composition and its component AS composition are shown. Figure 25A The average molecular weight in kDa of low-slip silk (LS) and AS82-AS89 is shown. Figure 25B Polydispersity (PDI) measurements are shown. Numerical data are presented in Table 9.

[0374] Figure 26 This is an SDS-polyacrylamide gel electrophoresis of low-slippery silk / modified polypeptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, with their respective silk compositions being: fraction 6 is AS82, fraction 7 is AS83, fraction 8 is AS84, fraction 9 is AS85, and fraction 10 is AS86.

[0375] Figure 27A and 27B Graph showing the self-assembly reaction of a low-sliding / modified peptide composition. A medium-sliding reaction was used as a positive control. Figure 27AThe kinetic parameters of gel formation during silk self-assembly are shown. The self-assembly parameters of the medium-sliding silk are: Amax is 0.6978 (Abs), SARF is 8.591, T0.5 is 3.361 h, and FSAF is 3.46 (Abs / min). Figure 27B This is a snapshot of the same self-assembly assay at a later time point, 18 days after assay setup. AS87, AS88, and AS89 show gel formation at this time point, which was already observed five days after the assay (LS, low sliding; MS, medium sliding).

[0376] Figures 28A-28C is a graph showing the characterization of low-slip silk compositions by dynamic light scattering. Low-slip silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the particle size of each silk composition. Figure 28A . Shown are the intensity particle size distributions measured for silk compositions AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89. Figure 28B Shown are the intensity size distributions measured for silk composition AS82, low slippery silk / modified peptide composition (LS), and medium slippery silk / modified peptide composition (MS). Figure 28C . Shown are the correlation plot functions of silk compositions AS82, AS83, AS84, AS85, AS86, AS87, AS88, AS89, low-slippery silk / modified peptide composition (LS), and medium-slippery silk / modified peptide composition (MS).

[0377] Figure 29Anion exchange chromatography (Q), hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) schemes for separating low-slippery / modified peptide compositions are shown. The low-slippery / modified peptide compositions are composed of a variety of peptide populations with a wide range of sizes and charges. Different low-slippery / modified peptide composition populations are isolated using Q-Sepharose anion exchange chromatography as the first step, Butyl ImpRes hydrophobic interaction resin as the second step, and HiLoad Superdex 200 size exclusion chromatography as the third purification step. The Q-Sepharose eluate is loaded onto a Butyl ImpRes (HIC) column, and the HIC-eluate is loaded onto a HiLoad Superdex 200 size exclusion chromatography, which results in the sorting of negatively charged silk compositions / modified peptides with hydrophobic characteristics sorted by size. The Q-Sepharose eluate contains negatively charged peptides of various sizes. Resolving these peptides by Butyl ImpRes results in the elution of high molecular weight, negatively charged, somewhat hydrophobic silk compositions / modified peptides. Smaller negatively charged peptides are washed as the flow-through and do not bind to the Butyl ImpRes column. The Q-HIC (eluate) is loaded onto a Superdex 200 and separated by size.

[0378] Figures 30A-30E Chromatograms of anion exchange chromatography, hydrophobic interaction chromatography, and subsequent size exclusion chromatography of a low-slipping / modified peptide composition. Figure 30A Anion exchange chromatography using a Q-Sepharose column is shown. Anion exchange chromatography separates the low-slip silk / modified peptide composition into an uncharged peptide population (flowthrough - light blue background) and elutes the negatively charged silk composition (eluate - light pink background). The light yellow background indicates that the column was washed with 50 mM Tris pH = 8.0 before eluting the charged peptide population. Figure 30B . Shown is the negatively charged eluent (Q-eluent) loaded onto a Butyl ImpRes column in the presence of 300 mM ammonium sulfate [(NH4)2SO4] to expose the hydrophobic domain of the silk peptide, which allows binding to the column. The highly charged peptide population does not bind to the column (effluent), highlighted in light blue. The column is washed until the OD280 decreases to approximately 100 units (light yellow). The bound silk peptide (Q-HIC (eluent)) is then eluted using 50 mM Tris, pH = 8.0 (light pink) without ammonium sulfate. Figure 30CThe Q-HIC (eluate) was further fractionated by size exclusion chromatography (SEC) using a Superdex 200 gel filtration column. The Q-HIC (eluate) fraction was passed through a column containing 50 mM Tris, 200 mM CaCl2, pH = 8.0. When the UV-280 absorbance began to increase, fractions were collected to separate the low-slip silk / modified peptide compositions by size. The relative elution volumes of silk compositions AS90 and AS94 are indicated on the chromatogram. Figure 30D The Q-HIC (flow-through) fraction was further fractionated by SEC using a Superdex 200 column following the same procedure as in (VC). The relative elution volumes of silk compositions AS95 and AS100 are indicated on the chromatogram. Figure 30E An overlay of chromatograms (VC) and (VD) is shown. The Q-HIC (eluate) fraction has a higher molecular weight range compared to the Q-HIC (flow-through) fraction, which elutes later in SEC and has a lower molecular weight range.

[0379] Figures 31A-31B is a graph showing analytical size exclusion chromatograms of a low slippery silk / modified silk composition and its component AS composition. Figure 31A The average molecular weight in kDa of low-slip silk (LS) and AS90-AS100 is shown. Figure 31B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 11.

[0380] Figures 32A-32B SDS polyacrylamide gel electrophoresis of low-slip / modified peptide compositions. Figure 32A .Q-HIC (eluate) SEC fractions. Figure 32B .Q-HIC (flow-through) SEC fractions. Lanes are indicated by fraction numbers in the order of elution from the Superdex 200 column, and their respective silk compositions are: Figure 32A In the , fraction 6 is AS90, fraction 7 is AS91, fraction 8 is AS92, fraction 9 is AS93, and fraction 10 is AS94. Figure 32B Of the 100, fraction 8 was AS95, fraction 9 was AS96, fraction 10 was AS97, fraction 11 was AS98, fraction 12 was AS99, and fraction 13 was AS100.

[0381] Figure 33The self-assembly reaction of a low-slipping silk / modified peptide combination is shown. The medium-slipping silk reaction was used as a positive control. Kinetic parameters of gel formation during silk self-assembly. Q-HIC (eluate) is the eluate fraction eluted from the Butyl ImpRes column before SEC purification; LS, low-slipping silk; MS, medium-slipping silk. Self-assembly parameters for medium-slipping silk: Amax of 0.6974 (Abs), SARF of 8.661, T0.5 of 3.834 h, and FSAF of 3.03 (Abs / min).

[0382] Figures 34A-34F Characterization of the low-slip silk composition by dynamic light scattering is shown. The low-slip silk and medium-slip silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the diameter particle size of each silk composition. Figure 34A Intensity diameter particle size distribution measured for silk composition AS90, Q-HIC (eluate) fraction (before sorting by SEC), low slip silk (LS) and medium slip silk (MS). Figure 34B .Correlation plot function of the silk composition presented in (34A). Figure 34C Intensity diameter particle size distribution measured for silk compositions AS90-AS94 derived from the Q-HIC (eluent)-SEC sorting process. Figure 34D .Correlation plot function of the silk composition presented in (34C). Figure 34E Intensity diameter particle size distribution measured for silk compositions AS95-AS100 derived from the Q-HIC (effluent)-SEC sorting process. Figure 34F .Correlation plot function of the silk composition presented in (34E).

[0383] Figure 35 A size exclusion chromatogram showing the separation of a mid-slippery / modified peptide composition is shown. Mid-slippery / modified peptide compositions consist of diverse peptide populations with a wide range of sizes. Using HiLoad Superdex 200 size exclusion chromatography, distinct mid-slippery / modified peptide composition populations can be separated.

[0384] Figure 36 The figure shows a chromatogram of a medium-smooth silk / modified peptide composition loaded on a Superdex 200 gel filtration column. The medium-smooth silk / modified peptide composition was loaded onto a Superdex 200 column and passed through a column containing 50 mM Tris, 200 mM CaCl2, pH 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the medium-smooth silk / modified peptide composition by size. The relative elution volumes of silk compositions AS107 and AS111 are indicated on the chromatogram.

[0385] Figures 37A-37B . Shown are analytical size exclusion chromatograms of the medium-slip silk / modified silk composition and its constituent AS compositions. Figure 37A The average molecular weight in kDa of medium silk (MS) and AS106-AS111 is shown. Figure 37B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 14.

[0386] Figure 38 Figure 2 is an SDS-polyacrylamide gel electrophoresis of the smooth silk / modified polypeptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, with their respective silk compositions being: fraction 6 is AS107, fraction 7 is AS108, fraction 8 is AS109, fraction 9 is AS110, and fraction 10 is AS111.

[0387] Figure 39 The self-assembly reaction of a medium-sliding silk / modified peptide composition is shown. Kinetic parameters for gel formation during silk self-assembly are shown. The red dashed line shows how the self-assembly parameters Amax, SARF, and T0.5 were calculated for unsorted medium-sliding silk (MS). These numerically calculated parameters for silk compositions AS106-AS111 can be found in Table 16. Low-sliding silk (LS) served as a negative control. LS, low-sliding silk; MS, medium-sliding silk.

[0388] Figures 40A-40B Figure 4 shows the characterization of the medium-sliding silk compositions by dynamic light scattering. The medium-sliding silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the particle size of each silk composition. Figure 40A . Intensity particle size distribution measured for silk compositions AS106, AS107, AS108, AS109, AS110, AS111 and medium-slip silk (MS). Figure 50B .Correlation functions for the silk compositions presented in (40A).

[0389] Figure 41 .Anion exchange chromatography and size exclusion chromatography schemes for separating mid-slide silk / modified peptide compositions are shown. Mid-slide silk / modified peptide compositions consist of diverse peptide populations with a wide range of sizes and charges. Different mid-slide silk / modified peptide composition populations were separated using Q-Sepharose anion exchange chromatography as the first step and HiLoad Superdex 200 size exclusion chromatography as the second purification step. The Q-Sepharose eluate was loaded onto a HiLoad Superdex 200 size exclusion chromatograph, which resulted in size sorting of negatively charged silk compositions / modified peptides.

[0390] Figures 42A-42B. is a chromatogram of anion exchange chromatography and subsequent size exclusion chromatography of the eluate (Q-eluate) of the mid-slippery silk / modified polypeptide composition. Figure 42A Anion exchange chromatography was performed using a Q-Sepharose column (Cytiva). Anion exchange chromatography separated the mid-slip silk / modified peptide composition into an uncharged peptide population (flowthrough - light blue background) and eluted negatively charged silk composition (eluate - light pink background). The light yellow background indicates that the column was washed with 50 mM Tris pH = 8.0 before eluting the charged peptide population. Figure 42B The negatively charged eluate (Q-eluate) was loaded onto a Superdex 200 column and passed through a column containing 50 mM Tris, 200 mM CaCl2, pH = 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the mid-slip silk / modified peptide compositions by size. The relative elution volumes of silk compositions AS101 and AS105 are indicated on the chromatogram.

[0391] Figures 43A-43B . Shown are analytical size exclusion chromatograms of the medium-slip silk / modified silk composition and its constituent AS compositions. Figure 43A The average molecular weight in kDa of medium silk (MS) and AS101-AS105 is shown. Figure 43B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 16.

[0392] Figure 44 A. is an SDS-polyacrylamide gel electrophoresis of the smooth silk / modified polypeptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, with their respective silk compositions being: fraction 6 is AS101, fraction 7 is AS102, fraction 8 is AS103, fraction 9 is AS104, and fraction 10 is AS105.

[0393] Figure 44B The self-assembly reaction of the medium-slip silk / modified peptide composition is shown. The low-slip silk reaction is used as a negative control. The kinetic parameters of gel formation during silk self-assembly are shown. The red dotted line is shown to illustrate the calculation of the Amax, SARF (self-assembly rate factor) and T0.5 parameters in Table 17.

[0394] Figures 45A-45C . is a graph showing the characterization of the medium-sliding silk compositions by dynamic light scattering. The medium-sliding silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro (Malvern) to estimate the diameter particle size of each silk composition. Figure 45A. Intensity diameter particle size distribution determined by intensity measurements for silk compositions AS101, AS102, AS103, AS104 and AS105. Figure 45B Intensity diameter particle size distribution determined by intensity measurements for silk compositions AS101, AS105 and medium-slip silk (MS) to emphasize the particle size difference between AS101 and AS105. Figure 45C . Correlation plot functions of silk compositions AS101, AS102, AS103, AS104, AS105 and medium sliding silk (MS).

[0395] Figure 46 is a graphical illustration of the values of the three molar mass moments (Mn, Mw, and Mz) as they relate to molar mass and the number of molecules per molar mass. This example applies to polydisperse samples; for monodisperse samples, Mn = Mw = Mz.

[0396] Figures 47A-47B It is an analytical SEC-MALS for low molecular weight silk, medium molecular weight silk and high molecular weight silk. Figure 47A . Weight average molecular weight in kDa of low molecular weight silk, medium molecular weight silk and high molecular weight silk. Figure 47B . Shows the polydispersity index (PDI) measurements for low molecular weight silk, medium molecular weight silk, and high molecular weight silk.

[0397] Figures 48A-48B Analytical SEC-MALS of low-, medium-, and high-molecular-weight silk produced with different process parameters and levels. Individual data points are shown, and the mean is represented by the height of the box. The bars cover one standard deviation. Figure 48A .The weight average molecular weight range of low molecular weight silk, medium molecular weight silk and high molecular weight silk. Figure 48B .PDI range of low molecular weight silk, medium molecular weight silk and high molecular weight silk.

[0398] Figures 49A-49B Analytical SEC-MALS of low slippery / modified silk compositions and component AS compositions separated by Q-SEC (Q-eluate). Figure 49A The average molecular weight in kDa of low-slip silk (LS) and AS77-AS81 is shown. Figure 49B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 24.

[0399] Figures 50A-50B Analytical SEC-MALS of low-slip silk / modified silk compositions and component AS compositions separated by SEC. Figure 50A The average molecular weight in kDa of low-slip silk (LS) and AS82-AS89 is shown. Figure 50BThe polydispersity (PDI) measurements are shown. The numerical data are presented in Table 25.

[0400] Figures 51A-51B Analytical SEC-MALS of low slippery / modified silk compositions and component AS compositions separated by Q-HIC-SEC (Q-HIC-eluate). Figure 51A The average molecular weight in kDa of low-slip silk (LS) and AS90-AS94 is shown. Figure 51B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 26.

[0401] Figures 52A-52B Analytical SEC-MALS of low slip / modified silk composition and component AS composition separated by Q-HIC-SEC (Q-HIC-flowthrough). Figure 52A The average molecular weight in kDa of low-slip silk (LS) and AS95-AS100 is shown. Figure 52B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 26.

[0402] Figures 53A-53B Analytical SEC-MALS of the mid-slip silk / modified silk composition and the component AS composition separated by Q-SEC (Q-flowthrough). Figure 53A The average molecular weight in kDa of medium silk (MS) and AS101-AS105 is shown. Figure 53B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 27.

[0403] Figures 54A-54B is the analysis of the mid-slip silk / modified silk composition and the component AS composition separated by SEC-MALS. Figure 54A The average molecular weight in kDa of medium silk (MS) and AS106-AS111 is shown. Figure 54B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 28.

[0404] Figure 55 TABLE 1 is a graph showing average TEWL values of barrier-reducing emulsions.

[0405] Figure 56 is a graph showing the effect of barrier restoring emulsion on wrinkles and photoaging.

[0406] Figure 57 is a graph showing the effect of barrier-restore emulsion on hyperpigmentation.

[0407] Figure 58 This is a graph showing the effect of barrier-restoring emulsion on wrinkles.

[0408] Figure 59 Graph showing the effect of barrier-restored emulsion on crow's feet wrinkles.

[0409] Figure 60 Graph showing the effect of barrier-restore lotion on crow's feet.

[0410] Figure 61 is a graph showing the effect of barrier restoring lotion on wrinkles in the puppet zone.

[0411] Figure 62 This is a graph showing the effect of barrier-restored emulsion on rough skin.

[0412] Figure 63 Graph showing the effect of barrier-restore lotion on overall wrinkles.

[0413] Figure 64 is a graph showing the effect of barrier-restore lotion on overall fine lines.

[0414] Figure 65 is a chart showing the effect of Barrier Restoring Lotion on the appearance of medium and deep lines and wrinkles.

[0415] Figure 66 Graph showing the effect of barrier-restore lotion on skin tone evenness.

[0416] Figure 67 is a graph showing the effect of barrier restoring lotion on skin texture.

[0417] Figure 68 Graph showing the effect of barrier-restore lotion on skin exfoliation.

[0418] Figures 69A-69C A sequence listing of the fibroin heavy chain is shown.

[0419] Figure 70 A sequence listing of the fibroin light chain is shown.

[0420] Figure 71 A sequence listing of a fibroin hexamer is shown.

[0421] Figure 72 is a graph showing the absorbency of different fabrics using laundry pods with activated filaments.

[0422] Figure 73 is a flow chart showing various embodiments for producing the silk fibroin fragments (SPF) of the present disclosure.

[0423] Figure 74 is a flow chart showing various parameters that can be modified during the process of producing the silk protein fragment solutions of the present disclosure during the extraction and solubilization steps.

[0424] Figure 75 is a graph showing the absorbency of Activated Silk™ with an octyl / octyl glucoside coating on various interlocking nylon fabrics; the unfinished nylon interlock fabrics were unable to absorb water due to poor absorbency; after coating the Activated Silk™ with the octyl / octyl glucoside coating, the absorbency of all nylon interlock fabrics increased significantly.

[0425] Figure 76 is a graph showing the absorbency of Active Silk™ with an octyl / octyl glucoside coating on various nylon fabrics in addition to the interlocking structure; the unfinished nylon fabrics did not absorb water or absorbed it poorly; after coating Active Silk™ with the octyl / octyl glucoside coating, the absorbency of all nylon fabrics increased significantly.

[0426] Figure 77 is a graph showing the unwashed water absorption curves produced by varying the concentration of polyoxyethylene (29) castor oil in the emulsifier mixture (thereby varying the HLB) before addition to the coating solution; in all samples, the silk concentration in the coating solution was 1 g / L.

[0427] Figure 78 is a graph showing the unwashed feel rating curves produced by varying the concentration of polyoxyethylene (29) castor oil in the emulsifier mixture (thereby varying the HLB) before addition to the coating solution; in all samples, the silk concentration in the coating solution was 1 g / L.

[0428] Figures 79A-79D Figure 2 shows the results of unwashed ( ) coatings produced by varying the concentrations of the emulsion mixture (polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan trioleate, polyoxyethylene (29) castor oil, and water in a ratio of 2:4:8:10) in the final coating solution. Figure 79A ), 5 washes( Figure 79B ), 10 washes( Figure 79C ) and 25 washes ( Figure 79D ) graph of moisture management data for the ® 1000 nanowires; in all samples, the silk concentration in the coating solution was 1 g / L.

[0429] Figures 80A-80D Figure 2 shows the results of unwashed ( ) coatings produced by varying the concentrations of the emulsion mixture (polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan trioleate, polyoxyethylene (29) castor oil, and water in a ratio of 2:4:8:10) in the final coating solution. Figure 80A ), 5 washes( Figure 80B ), 10 washes( Figure 80C) and 25 washes ( Figure 80D ) of the feel grade results; 1 is the best feel grade score and 8 is the worst feel grade score; in all samples, the silk concentration in the solution was 1g / L.

[0430] Figures 81A-81D The results show that the unwashed ( Figure 81A ), 5 washes( Figure 81B ), 10 washes( Figure 81C ) and 25 washes ( Figure 81D ) of the feel rating results; 1 is the best rating and 8 is the worst rating

[0431] Figures 82A-82D The results show that the unwashed ( Figure 82A ), 5 washes( Figure 82B ), 10 washes( Figure 82C ) and 25 washes ( Figure 82D ) of the water management results.

[0432] Figures 83A-83D is a graph showing a UV / Vis quantitative experiment of a fabric coated with low molecular weight active silk and polyoxyethylene (20) monooleate solution. Figure 83A : Graph showing the percentage of silk loss after five washes relative to the fiber surface area. Figure 83B : Graph showing the quantitative mass of silk on the fabric after coating relative to the fiber surface area. Figure 83C : Graph showing the percentage of silk loss after five washes relative to fabric type. Figure 83D : Graph showing the quantitative mass of silk on each fabric before and after five washes according to fabric type.

[0433] Figure 84 This is a graph showing a UV / Vis quantification experiment of a fabric coated with a low molecular weight reactive yarn and a solution of polyoxyethylene (20) monooleate. The graph shows the quantitative mass of the yarn on the fabric after coating relative to the fabric mass in grams per square meter (GSM). This mass depends on the weave type, fiber content, and filament denier.

[0434] Figures 85A-85C Includes a series of diagrams showing the properties of unfinished heavyweight double-weave nylon fabric ( Figure 85A ), heavy double-sided woven nylon fabric with active yarn finishing ( Figure 85B ) and Archroma RPU wetting agent finished heavy double woven nylon fabric ( Figure 85C) is a potentiometric titration curve of the charge density measured at pH 5. Each fabric has a Figures 85A-85C , left) and five washes ( Figures 85A-85C , right). The change in charge density at pH 5 after washing is shown as ΔC.

[0435] Figure 86 Shown is the experimental setup for Example 18. Fabric samples were prepared and washed five times in a front-loading washing machine, and the surface charge was measured as the charge difference before and after washing.

[0436] Figure 87 is a diagram showing the location and description of the zeta potential of a surface.

[0437] Figure 88 is a graph showing the change in zeta potential of five different fabric types and three different finish types before and after five washes.

[0438] Figure 89 is a graph showing the effect of effective surface charge on the surrounding pH due to proton and hydroxide surface adsorption.

[0439] Figure 90 is a graph showing the change in charge density of five different fabric types before and after five washes at pH 5. The control section represents unfinished fabric and the AS 320 section represents silk finished fabric.

[0440] Figure 91 is a graph showing the UV / Vis spectrum of the synthetic silver wire nanoparticles tested in Example 19.

[0441] Figure 92 is a graph showing the absorbance from T=0 to T=10 for unfinished dark blue DC98-IA pretreated with fixing agent only and for AS-320 coated dark blue DC98-IA pretreated with cationic fixing agent.

[0442] Figure 93 is a graph showing the absorbance from T=0 to T=10 of unfinished black DC95-FA pretreated with fixing agent only and AS-320 coated black DC95-FA pretreated with cationic fixing agent.

[0443] Figure 94 is a graph showing the absorbance of unfinished and AS-320 coated (15 g / L, with and without pretreatment with HT-232H fixing agent) 17120513 black from T=0 to T=10.

[0444] Figure 95is a graph showing the absorbance of unfinished and AS-320 coated (15 g / L, with and without pretreatment with HT-232H fixative) 17120511 blue from T=0 to T=10.

[0445] Figure 96 is a graph showing the improvement in Investigator Global Assessment (IGA) after using the body wash.

[0446] Figure 97 This is a graph showing the improvement in redness after using the body wash.

[0447] Figure 98 It is a graph showing the improvement of eczema count after using the body wash.

[0448] Figure 99 Graph showing improvement in stratum corneum moisture level after using body wash.

[0449] Figure 100 is a graph showing the improvement in individual TEWL after using the body wash.

[0450] Figure 101 This is a graph showing the responses to the self-perception questionnaire after using the body wash.

[0451] Figure 102 This is a graph showing the responses to the self-perception questionnaire after using the body wash.

[0452] Figure 103 Three chromatographic principles of silk sorting are shown.

[0453] Figure 104 Size exclusion chromatography of silk sorting is shown.

[0454] Figure 105 Anion exchange chromatography and subsequent size exclusion chromatography of silk sorting are shown.

[0455] Figure 106 Anion exchange chromatography followed by hydrophobic interaction chromatography and size exclusion chromatography is shown.

[0456] Figure 107 This is a diagram summarizing the three pipelines for silk sorting.

[0457] Figure 108 It is a characterization method for silk sorting.

[0458] Figure 109 Size exclusion chromatography of low-slip silk is shown.

[0459] Figure 110 An example of silk composition characterization is shown: size exclusion chromatography of low slip silk.

[0460] Figure 111An example of silk composition characterization is shown: size exclusion chromatography with dynamic light scattering of low-slip silk.

[0461] Figure 112 is a graph of the Z-average values of a composition of polypeptides comprising low-slippery / modified polypeptides.

[0462] Figure 113 Molecular weight and polydispersity determined by HPLC are shown.

[0463] Figure 114 Silk sorting size exclusion chromatography showing low-slip silk. Over time, the lower molecular weight fraction slowly self-assembles.

[0464] Figure 115 is a graph including assays used to characterize the silk fraction.

[0465] Figure 116 is a comparison of molecular weight and polydispersity determinations performed by the two methods.

[0466] Figure 117 A graph is shown presenting the data from Table 70 and Table 71. Data are shown with standard deviation. "Nanoclay" refers to Elementis Bentone Hydroclay.

[0467] Figure 118 This is a diagram of a typical bentonite clay structure.

[0468] Figure 119 Shown is an SEM image of a cross-section of a film cast from Elementis Bentone Hydroclay 2001. Highly ordered stacking of clay layers is visible.

[0469] Figure 120 is an SEM image of the cross section of a film cast with pure RSF.

[0470] Figure 121 This is an SEM image of a cross-section of a 1:1 RSF / 2001 film cast under neutral conditions (pH approximately 7.0). The layered structure of the clay is preserved.

[0471] Figure 122 This is an SEM image of a cross-section of a 1:1 RSF / 2001 film cast under acidic conditions (pH approximately 3.5). Note the ribbon-like structure.

[0472] Figure 123 The enhanced diffusion pathways created by the RSF / nanoclay composite are shown.

[0473] Figure 124FTIR scan of the amide I region of RSF / 2001 films cast under neutral conditions. The nanoclay concentration was varied from 0% (red) to 70% (yellow). As the nanoclay content increased, the amide I peak shifted to the left, away from the β-sheet region. DETAILED DESCRIPTION

[0474] In some embodiments, the compositions of the present disclosure include peptide compositions selected from compositions #1001 to #2450 having a weight average molecular weight selected from about 1 kDa to about 145 kDa, and a polydispersity selected from 1 to about 5 (including but not limited to a polydispersity of 1), 1 to about 1.5 (including but not limited to a polydispersity of 1), about 1.5 to about 2, about 1.5 to about 3, about 2 to about 2.5, about 2.5 to about 3, about 3 to about 3.5, about 3.5 to about 4, about 4 to about 4.5, and about 4.5 to about 5.

[0475] Methods for preparing silk fibroin or silk fibroin fragments and their use in various fields are known and described, for example, in U.S. Patents 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369 and 10,166,177, 10,287,728 and 10,301,768, all of which are incorporated herein by reference in their entirety. Raw silk from the silkworm (Bombyx mori) consists of two main proteins: fibroin (about 75%) and sericin (about 25%). Fiber fibroin is a fibrous protein with a semi-crystalline structure that provides stiffness and strength. The term "fibroin" as used herein refers to fibers from the cocoon of the silkworm Bombyx mori having a weight-average molecular weight of about 370,000 Da. Crude silkworm fiber consists of double strands of fibroin. The adhesive material that binds these twin fibers together is sericin. Silk fibroin is composed of heavy chains (H chains) with a weight-average molecular weight of approximately 350,000 Da and light chains (L chains) with a weight-average molecular weight of approximately 25,000 Da. Silk fibroin is an amphiphilic polymer with a large hydrophobic domain (of high molecular weight) that constitutes the majority of the polymer. The hydrophobic region is interrupted by small hydrophilic spacers, and the N- and C-termini of the chain are also highly hydrophilic. The hydrophobic domain of the H chain contains a repeating hexapeptide sequence of Gly-Ala-Gly-Ala-Gly-Ser and repeats of the Gly-Ala / Ser / Tyr dipeptide, which can form stable anti-parallel-sheet crystallites. The amino acid sequence of the L chain is non-repeating, making it more hydrophilic and relatively elastic. The alternating arrangement of hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) segments in the silk fibroin molecule allows for self-assembly of the fibroin molecule.

[0476] The term "fibroin" as used herein includes silk fibroin and insect or spider silk proteins. In one embodiment, the silk fibroin is obtained from the silkworm, Bombyx mori. Raw silk from the silkworm is composed of two main proteins: fibroin (about 75%) and sericin (about 25%). The term "fibroin" as used herein refers to fibers from the cocoon of the silkworm, Bombyx mori, having a weight-average molecular weight of about 370,000 Da. Methods for preparing silk fibroin fragments and / or their compositions are known and are described, for example, in U.S. Patents 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369 and 10,166,177.

[0477] Recombinant silk is described in the patents and patent applications incorporated herein by reference: US 2004590196, US 7,754,851, US 2007654470, US 7,951,908, US 2010785960, US 8,034,897, US 20090263430, US 2008226854, US 20090123967, US 2005712095, US 2007991037, US 20090162896, US200885266, US 8,372,436, US 2007989907, US 2009267596, US 2010319542, US2009265344, US 2012684607, US 2004583227, US 8,030,024, US 2006643569, US 7,868,146, US 2007991916, US 8,097,583, US 2006643200, US 8,729,238, US 8,877,903, US20190062557, US 20160280960, US 20110201783, US 2008991916, US 2011986662, US2012697729, US 20150328363、US 9,034,816, US 20130172478, US 9,217,017, US20170202995, US 8,721,991, US 2008227498, US 9,233,067, US 8,288,512, US2008161364, US 7,148,039, US1999247806, US 2001861597, US 2004887100, US 9,481,719, US 8,765,688, US 200880705, US 2010809102, US 8,367,803, US 2010664902、US 7,569,660, US1999138833, US 2000591632, US 20120065126, US 20100278882, US2008161352, US 20100015070, US 2009513709, US 20090194317, US 2004559286, US200589551, US 2008187824, US 20050266242, US 20050227322 and US 20044418.

[0478] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US 20190062557, US 20150284565, US 20130225476, US 20130172478, US 20130136779, US 20130109762, US 20120252294, US 20110230911, US 20110201783, US 20100298877, US 10,478,520, US 10,253,213, US 10,072,152, US 9,233,067, US 9,217,017, US 9,034,816, US 8,877,903, US 8,729,238, US 8,721,991, US 8,097,583, US 8,034,897, US 8,030,024, US 7,951,908, US 7,868,146 and US 7,754,851.

[0479] The recombinant silk proteins and / or methods described herein may include one or more of the following: described above or in U.S. Patents 8,173,772, 8,278,416, 8,618,255, 8,642,734, 8,691,581, 8,729,235, 9,115,204, 9,157,070, 9,309,299, 9,644,012, 9,708,376, 9,051,453, 9,617,315, 9,968,682, 9,689,089, 9,732,125, 9,856,308, 9,926,348, 10,065,997, 10,316,069, and 10,329,332;and U.S. Patent Publications 2009 / 0226969, 2011 / 0281273, 2012 / 0041177, 2013 / 0065278, 2013 / 0115698, 2013 / 0316376, 2014 / 0058066, 2014 / 0079674, 2014 / 0245923, 2015 / 0087046, 2015 / 0119554, 2015 / 0141618, 2015 / 0291673, 2015 / 0291674, 2015 / 02395 87, 2015 / 0344542, 2015 / 0361144, 2015 / 0374833, 2015 / 0376247, 2016 / 0024464, 2017 / 0066804, 2017 / 0066805, 2015 / 0293076, 2016 / 0222174, 2017 / 0283474, 2017 / 0088675, 2019 / 0135880, 2015 / 0329587, 2019 / 0040109, 2019 / 0135881, 2 019 / 0177363, 2019 / 0225646, 2019 / 0233481, 2019 / 0031842, 2018 / 0355120, 2019 / 0186050, 2019 / 0002644, 2020 / 0031887, 2018 / 0273590, 20191 / 094403, 2019 / 0031843, 2018 / 0251501, 2017 / 0066805, 2018 / 0127553, 2019 / 0329526, 2020 / 0031886, 2018 / 0080147, 2019 / 0352349, 2020 / 0043085, 2019 / 0144819, 2019 / 0228449, 2019 / 0340666, 2020 / 0000091, 2019 / 0194710, 2019 / 0151505, 2018 / 0265555, 2019 / 0352330, 2019 / 0248847, and 2019 / 0378191 (which are incorporated herein by reference in their entireties).

[0480] As used herein, the term "substantially free of inorganic residues" means that the composition exhibits 0.1% (w / w) or less residues. In one embodiment, substantially free of inorganic residues refers to a composition that exhibits 0.05% (w / w) or less residues. In one embodiment, substantially free of inorganic residues refers to a composition that exhibits 0.01% (w / w) or less residues. In one embodiment, the amount of inorganic residues is from 0 ppm ("not detectable" or "ND") to 1000 ppm. In one embodiment, the amount of inorganic residues is from ND to about 500 ppm. In one embodiment, the amount of inorganic residues is from ND to about 400 ppm. In one embodiment, the amount of inorganic residues is from ND to about 300 ppm. In one embodiment, the amount of inorganic residues is from ND to about 200 ppm. In one embodiment, the amount of inorganic residues is from ND to about 100 ppm. In one embodiment, the amount of inorganic residues is from 10 ppm to 1000 ppm.

[0481] As used herein, the term "substantially free of organic residues" means that the composition exhibits 0.1% (w / w) or less residues, and in one embodiment, substantially free of organic residues means that the composition exhibits 0.05% (w / w) or less residues. In one embodiment, substantially free of organic residues means that the composition exhibits 0.01% (w / w) or less residues. In one embodiment, the amount of organic residues is from 0 ppm ("not detectable" or "ND") to 1000 ppm. In one embodiment, the amount of organic residues is from ND to about 500 ppm. In one embodiment, the amount of organic residues is from ND to about 400 ppm. In one embodiment, the amount of organic residues is from ND to about 300 ppm. In one embodiment, the amount of organic residues is from ND to about 200 ppm. In one embodiment, the amount of organic residues is from ND to about 100 ppm. In one embodiment, the amount of organic residues is from 10 ppm to 1000 ppm.

[0482] The compositions of the present disclosure exhibit "biocompatibility", meaning that the compositions are compatible with living tissues or life systems due to being non-toxic, harmless or having no physiological reactivity and not causing immune rejection. Such biocompatibility can be demonstrated by participants applying the compositions of the present disclosure topically on their skin for an extended period of time. In one embodiment, the extended period of time is about 3 days. In one embodiment, the extended period of time is about 7 days, in one embodiment, the extended period of time is about 14 days, in one embodiment, the extended period of time is about 21 days. In one embodiment, the extended period of time is about 30 days. In one embodiment, the extended period of time is selected from the group consisting of: about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months and indefinite.

[0483] The compositions of the present disclosure can be "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by having participants topically apply the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period of time is about 3 days. In one embodiment, the extended period of time is about 7 days. In one embodiment, the extended period of time is about 14 days. In one embodiment, the extended period of time is about 21 days. In one embodiment, the extended period of time is about 30 days. In one embodiment, the extended period of time is selected from the group consisting of: about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0484] In one embodiment, the peptide composition of the present disclosure has an undetectable level of LiBr residues. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is 10ppm to 1000ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is 10ppm to 300ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 25ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 50ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 75ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 100ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 200ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 300ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 400ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 500ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 600ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 700 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 800 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 900 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 1000 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 500 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 450 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 400 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 350 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 300 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 250 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 200 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from undetectable to 150 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from undetectable to 100 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from 100 ppm to 200 ppm.In one embodiment, the amount of LiBr residual in the composition of the present disclosure is 200 ppm to 300 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is 300 ppm to 400 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is 400 ppm to 500 ppm.

[0485] In one embodiment, the peptide composition of the present disclosure has an undetectable level of Na2CO3 residuals. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 100ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 200ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 300ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 400ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 500ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 600ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 700ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 800ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 900ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 1000ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 500ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 450ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 400ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 350ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 300ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 250ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 200ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 150ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 100ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is 100ppm to 200ppm. In one embodiment, the amount of Na2CO3 residual in the composition of the present disclosure is 200 ppm to 300 ppm. In one embodiment, the amount of Na2CO3 residual in the composition of the present disclosure is 300 ppm to 400 ppm. In one embodiment, the amount of Na2CO3 residual in the composition of the present disclosure is 400 ppm to 500 ppm.

[0486] As used herein, the term "about" when referring to a number or numerical range means that the number or numerical range is included together with the number or numerical range within the experimental variability, or within the statistical experimental error of the number or numerical range, wherein the variability or error is from 0% to 15%, or from 0% to 10%, or from 0% to 5% of the number or numerical range.

[0487] Silk protein or its fragment, silk solution or mixture (for example, SPF or SFS solution or mixture) etc. can be prepared according to the method described in U.S. Patent Nos. 9,187,538, 9,522,107, 9,522,108, 9,511,012, 9,517,191, 9,545,369 and 10,166,177 and U.S. Patent Publication Nos. 2016 / 0222579 and 2016 / 0281294 and International Patent Publication Nos. WO 2016 / 090055 and WO2017 / 011679 (which are incorporated herein by reference in their entirety). Methods of using silk fibroin or silk fibroin fragments in coating applications are known and described in, for example, U.S. Patent Nos. 10,287,728 and 10,301,768.

[0488] The present disclosure provides articles comprising coated fabrics, wherein the coating comprises a surfactant and / or emulsifier system and silk fibroin fragments, including modified silk fragments disclosed herein, such as, but not limited to, silk fibroin peptides and / or protein fragments, wherein at least one of the amino acids is modified, substituted or replaced as disclosed herein, and methods of making such articles.

[0489] Silk is a natural polymer produced by various insects and spiders. Silk produced by the silkworm Bombyx mori (Bombyx mori) consists of a core protein, fibroin, and a gelatinous coating composed of the non-silk protein, sericin. Fibroin is an FDA-approved, edible, non-toxic, and relatively inexpensive protein derived from silkworm cocoons. The structure and content of amino acids in fibroin closely resemble those in human tissue.

[0490] Methods for preparing silk fibroin fragments are known and are described, for example, in U.S. Patents 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177. Methods for using silk fibroin or silk fibroin fragments in coating applications (including coating applications of animal hair) are known and are described, for example, in U.S. Patent Application Publication Nos. 20160222579 and 20160281294. Compositions and methods for using silk fibroin or silk fibroin fragments in cosmetic applications are known and are described, for example, in U.S. Patent Application Publication Nos. 20180280274 and 20180008522 and International Patent Application Publication No. WO 2019005848. All publications cited herein are incorporated herein by reference in their entirety.

[0491] The latest progress in silk material technology includes the emergence of top-down and bottom-up silk cocoon engineering, especially the aqueous solution of cocoon regeneration into silk, and the use of genetic engineering to produce recombinant silk with a molecularly determined composition (Tran et al., A review of the emerging role of silk for the treatment of the eye, Pharm. Res., 2018, Vol. 35, pp. 1-16). In recent years, it has been reported that fibroin has been used in eye tissue reconstruction, corneal tissue engineering and ocular surface repair due to its biocompatibility, adjustable properties and transparency. Silk membranes have been found to support corneal cell growth and form stratified epithelial cell sheets that are equivalent to the base of amniotic membrane (Lawrence et al., Silk film biomaterials for cornea tissue engineering, Biomaterials, 2009; 30(7): 1299-308; Harkin et al., Silk fibroin in ocular tissue reconstruction, Biomaterials, 2011; Chirtla et al., Bombyx mori silk fibroin membranes as potential substrata forepithelial constructs used in the management of ocular surface disorders. Tissue Engineering Pan A, 2008; 14(7): 1203-11.). It has been demonstrated that fibroin and hydrolyzed peptide fragments can inhibit the transcription and upstream activation of NF-κB protein subunits and proinflammatory molecules, which are normally controlled by NF-κB (Hayden et al., Cell Research, 2011. 21(2):223-244; Chon et al., International Journal of Molecular Medicine, 2012. 30(5):1203-1210; Kim et al., J. Neurosurg., 2011. 114(2):485-90; J. Microbiol. Biotechnol., 2012. 22(4):494-500).

[0492] definition

[0493] As used in the foregoing part of this specification and throughout the rest of this specification, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0494] Unless otherwise specified, all percentages, parts, and ratios are based upon the total weight of the eye care compositions of the present disclosure. All such weights as they relate to listed ingredients are based on the active level and, therefore, do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified. The term "weight percent" may be expressed herein as "wt %" or % w / w.

[0495] As used herein, the terms "a," "an," or "the" are generally construed to cover both the singular and the plural.

[0496] As used herein, the term "about" generally refers to a particular value that is within an acceptable error range as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean zero variation, as well as a range of ±20 percent, ±10 percent, or ±5 percent of a given value.

[0497] For example, as used herein, the term "dermatologically acceptable carrier" means a carrier that is suitable for use in contact with mammalian keratinous tissue without causing any adverse reactions (such as excessive toxicity, incompatibility, instability, allergic reactions). Dermatologically acceptable carriers may include, but are not limited to, water, liquid or solid emollients, humectants, solvents, and the like.

[0498] As used herein, the term "hydrophile-lipophile balance" (HLB) of a surfactant and / or emulsifier is a measure of its degree of hydrophilicity or hydrophobicity, as determined by calculating the values for different regions of the molecule, as determined by the Griffin method: HLB = 20*M h / M, where M his the molecular weight of the hydrophilic portion of the surfactant and / or emulsifier, and M is the molecular weight of the entire surfactant and / or emulsifier molecule. The results are in the range of 0 to 20. An HLB value of 0 corresponds to a completely lipophilic molecule, and an HLB value of 20 corresponds to a completely hydrophilic molecule. The HLB value can be used to predict the surfactant and / or emulsifier properties of a molecule: HLB < 10: fat-soluble (water-insoluble), HLB > 10: water-soluble (fat-insoluble), HLB = 1-3: defoamers, 3-6: W / O (water-in-oil) emulsifiers, 7-9: wetting and spreading agents, 8-16: O / W (oil-in-water) emulsifiers, 13-16: detergents, and 16-18: solubilizers or hydrotropes.

[0499] As used herein, "average weight average molecular weight" refers to the average of two or more values of weight average molecular weight of fibroin or fragments thereof of the same composition, the two or more values being determined by two or more separate experimental readings.

[0500] As used herein, the term polymer "polydispersity (PD)" is generally used as a measure of the breadth of the molecular weight distribution of a polymer and is defined by the following formula: Polydispersity

[0501] As used herein, the term "substantially uniform" may refer to fibroin-based protein fragments that are distributed in a normal distribution about an identified molecular weight. As used herein, the term "substantially uniform" may refer to a uniform distribution of components or additives, such as fibroin fragments, dermatologically acceptable carriers, etc., throughout the composition of the present disclosure.

[0502] As used herein, the terms "silk fibroin peptide," "silk fibroin protein fragment," and "silk fibroin fragment" are used interchangeably. Where molecular size is an important parameter, the molecular weight or the number of amino acid units is defined.

[0503] As used herein, the term "fast-dissolving solid form" refers to fast-dissolving solid forms including lyophilized forms (filter cakes, flakes, films) and compressed tablets.

[0504] As used herein, the term "peptide" or "protein" refers to a chain of amino acids linked together by peptide bonds (also called amide bonds). The fundamental differentiating factors between proteins and peptides are size and structure. Peptides are smaller than proteins. Traditionally, peptides are defined as molecules consisting of 2 to 50 amino acids, while proteins are composed of 50 or more amino acids. Additionally, the structure of peptides is often less well-defined than that of proteins, which can adopt complex conformations known as secondary, tertiary, and quaternary structures.

[0505] As used herein, the term "fibroin" or "silk protein" refers to a class of structural proteins produced by certain silk-producing spider and insect species (see the definition provided in WIPO Pearl - WIPO's Multilingual Terminology Portal database https: / / wipopearl.wipo.int / en / linguistic). Fibroin can include silkworm fibroin, insect or spider silk proteins (e.g., spidroin), recombinant spider proteins, silk proteins present in other spider silk types, such as tubular silk proteins (TuSP), flagellar silk proteins, minor ampullate silk proteins, alveolar silk proteins, piriform silk proteins, polymeric sericin), silkworm fibroin produced by genetically modified silkworms, or recombinant silkworm fibroin.

[0506] As used herein, the term "fibroin" refers to silkworm fibroin, silk fibroin produced by genetically modified silkworms, or recombinant silkworm fibroin (see (1) Narayan, ed., Encyclopedia of Biomedical Engineering, Vol. 2, Elsevier, 2019; (2) Kobayashi et al., ed., Encyclopedia of Polymeric Nanomaterials, Springer, 2014, https: / / link.springer.com / referenceworkentry / 10.1007%2F978-3-642-36199-9_323-1). In one embodiment, the silk fibroin is obtained from Bombyx mori.

[0507] As used herein, the term "solid solution" refers to an active agent that is molecularly dissolved in a solid excipient matrix, such as a hydrophobic polymer, wherein the active agent is miscible with the polymer matrix excipient.

[0508] As used herein, the term "solid dispersion" refers to an active agent dispersed as crystalline or amorphous particles, wherein the active agent is dispersed in an amorphous polymer and randomly distributed among the polymer matrix excipients.

[0509] As used herein, the term "substantially homogeneous" may refer to a distribution of fibroin-based protein fragments in a normal distribution with respect to an identified molecular weight. As used herein, the term "substantially homogeneous" may also refer to a uniform distribution of components or additives (e.g., fibroin-based protein fragments, dermatologically acceptable carriers, etc.) throughout the silk eye care composition.

[0510] As used herein, the term "surface tension" refers to the tendency of a fluid's surface to contract to the smallest possible surface area. At a liquid-air interface, surface tension arises because the liquid's molecules are more attracted to each other (due to cohesion) than the molecules in air are to each other (due to adhesion). The net effect is an inward force on the liquid's surface, which causes the liquid to behave as if its surface were covered by a stretched elastic film. Water has a higher surface tension (72.8 mN / m at 20°C) than most other liquids due to the relatively high attraction between its molecules through a network of hydrogen bonds.

[0511] SPF definition and properties

[0512] As used herein, "silk protein fragment" (SPF) includes, but is not limited to, one or more of the following: "fibroin fragment" as defined herein; "recombinant silk fragment" as defined herein; "spider silk fragment" as defined herein; "fibroin-like protein fragment" as defined herein; "chemically modified silk fragment" as defined herein; "sericin or sericin fragment" as defined herein; and / or modified silk fragments disclosed herein, such as, but not limited to, fibroin peptides and / or protein fragments, wherein at least one of the amino acids is modified, substituted, or replaced as disclosed herein. The SPF may have any molecular weight value or range described herein, and any polydispersity value or range described herein.

[0513] SPF molecular weight and polydispersity

[0514] In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 1 kDa to about 5 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 5 kDa to about 10 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 10 kDa to about 15 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 15 kDa to about 20 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 14 kDa to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 20 kDa to about 25 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 25 kDa to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 30 kDa to about 35 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 35 kDa to about 40 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 39 kDa to about 54 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 40 kDa to about 45 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 45 kDa to about 50 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 50 kDa to about 55 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 55 kDa to about 60 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 60 kDa to about 65 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 65 kDa to about 70 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 70 kDa to about 75 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 75 kDa to about 80 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 80 kDa to about 85 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 85 kDa to about 90 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 90 kDa to about 95 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 95 kDa to about 100 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 100 kDa to about 105 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 105 kDa to about 110 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 110 kDa to about 115 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 115 kDa to about 120 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 120 kDa to about 125 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 125 kDa to about 130 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 130 kDa to about 135 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 135 kDa to about 140 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 140 kDa to about 145 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 145 kDa to about 150 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 150 kDa to about 155 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 155 kDa to about 160 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 160 kDa to about 165 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 165 kDa to about 170 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 170 kDa to about 175 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 175 kDa to about 180 kDa.

[0515] In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 180 kDa to about 185 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 185 kDa to about 190 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 190 kDa to about 195 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 195 kDa to about 200 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 200 kDa to about 205 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 205 kDa to about 210 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 210 kDa to about 215 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 215 kDa to about 220 kDa.

[0516] In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 220 kDa to about 225 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 225 kDa to about 230 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 230 kDa to about 235 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 235 kDa to about 240 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 240 kDa to about 245 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 245 kDa to about 250 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 250 kDa to about 255 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 255 kDa to about 260 kDa.

[0517] In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 260 kDa to about 265 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 265 kDa to about 270 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 270 kDa to about 275 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 275 kDa to about 280 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 280 kDa to about 285 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 285 kDa to about 290 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 290 kDa to about 295 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 295 kDa to about 300 kDa.

[0518] In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 300 kDa to about 305 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 305 kDa to about 310 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 310 kDa to about 315 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 315 kDa to about 320 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 320 kDa to about 325 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 325 kDa to about 330 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 330 kDa to about 335 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 335 kDa to about 340 kDa.

[0519] In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 340 kDa to about 345 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 345 kDa to about 350 kDa.

[0520] In some embodiments, the compositions of the present disclosure include SPF compositions selected from compositions #1001 to #2450 having a weight average molecular weight selected from about 1 kDa to about 145 kDa, and a polydispersity selected from 1 to about 5 (including but not limited to a polydispersity of 1), 1 to about 1.5 (including but not limited to a polydispersity of 1), about 1.5 to about 2, about 1.5 to about 3, about 2 to about 2.5, about 2.5 to about 3, about 3 to about 3.5, about 3.5 to about 4, about 4 to about 4.5, and about 4.5 to about 5:

[0521]

[0522]

[0523]

[0524]

[0525]

[0526] In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from 1 to about 5.0, including but not limited to a polydispersity of 1. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 1.5 to about 3.0. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from 1 to about 1.5, including but not limited to a polydispersity of 1. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 1.5 to about 2.0. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 2.0 to about 2.5. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 2.5 to about 3.0.

[0527] In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 3.0 to about 3.5. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 3.5 to about 4.0. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 4.0 to about 4.5. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 4.5 to about 5.0.

[0528] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.9.In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 5.0.

[0529] Silk fibroin fragments

[0530] Methods for preparing silk fibroin or silk fibroin fragments and their use in various fields are known and described, for example, in U.S. Patents 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369 and 10,166,177, 10,287,728 and 10,301,768, all of which are incorporated herein by reference in their entirety. Raw silk from the silkworm (Bombyx mori) consists of two main proteins: fibroin (about 75%) and sericin (about 25%). Fiber fibroin is a fibrous protein with a semi-crystalline structure that provides stiffness and strength. The term "fibroin" as used herein refers to fibers from the cocoon of the silkworm Bombyx mori having a weight-average molecular weight of about 370,000 Da. Crude silkworm fiber consists of double strands of fibroin. The adhesive material that binds these twin fibers together is sericin. Silk fibroin is composed of heavy chains (H chains) with a weight-average molecular weight of approximately 350,000 Da and light chains (L chains) with a weight-average molecular weight of approximately 25,000 Da. Silk fibroin is an amphiphilic polymer with a large hydrophobic domain (of high molecular weight) that constitutes the majority of the polymer. The hydrophobic region is interrupted by small hydrophilic spacers, and the N- and C-termini of the chain are also highly hydrophilic. The hydrophobic domain of the H chain contains a repeating hexapeptide sequence of Gly-Ala-Gly-Ala-Gly-Ser and repeats of the Gly-Ala / Ser / Tyr dipeptide, which can form stable anti-parallel-sheet crystallites. The amino acid sequence of the L chain is non-repeating, making it more hydrophilic and relatively elastic. The alternating arrangement of hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) segments in the silk fibroin molecule allows for self-assembly of the fibroin molecule.

[0531] Provided herein are methods for producing pure and highly scalable solutions of silk fibroin fragment mixtures that can be used in a variety of applications across multiple industries. Without wishing to be bound by any particular theory, it is believed that these methods are equally applicable to the fragmentation of any SPF described herein, including but not limited to recombinant silk proteins, and fragmentation of silk-like or silk fibroin-like proteins.

[0532] The term "fibroin" as used herein includes silk fibroin and insect or spider silk proteins. In one embodiment, the silk fibroin is obtained from the silkworm, Bombyx mori. Raw silk from the silkworm is composed of two major proteins: fibroin (about 75%) and sericin (about 25%). Fibroin is a fibrous protein with a semi-crystalline structure that provides stiffness and strength. The term "fibroin" as used herein refers to fibers from the cocoon of the silkworm, Bombyx mori, having a weight average molecular weight of about 370,000 Da. Converting these insoluble fibroin fibrils into water-soluble fibroin fragments requires the addition of concentrated neutral salts (e.g., 8-10 M lithium bromide), which interfere with the intermolecular and intramolecular ionic and hydrogen bonding that originally makes the silk fibroin insoluble in water. Methods of preparing fibroin fragments and / or compositions thereof are known and described, for example, in US Patents 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177.

[0533] Figure 73 is a flow chart illustrating a general embodiment for producing some embodiments of the present disclosure. It should be understood that not all illustrated steps are necessary to prepare all silk solutions of the present disclosure. Figure 74 As shown in step A, cocoons (heat-treated or not), silk fibers, silk powder, spider silk or recombinant spider silk can be used as the silk source.

[0534] Figure 74 is a flow chart showing a general embodiment of various parameters that can be modified during the process of producing the silk protein fragment solutions of the present disclosure during the extraction and solubilization steps.

[0535] In some embodiments specifically disclosed herein, the molecular weight of the fibroin fragment composition is determined using high pressure liquid chromatography (HPLC) with a refractive index detector (RID), and the polydispersity is calculated using Cirrus GPC Online GPC / SEC software version 3.3 (Agilent). In some embodiments specifically disclosed herein, the molecular weight of the fibroin fragment composition is determined using a SEC-MALS method.

[0536] Data Analysis and Calculations - Calculation of Average Molecular Weight Using Cirrus Software

[0537] Upload the chromatographic data files of the standard and analytical samples to the Cirrus SEC data collection and molecular weight analysis software. Calculate the weight average molecular weight (M) of each sample injected. w ), number average molecular weight (M n ), peak average molecular weight (M p ) and polydispersity.

[0538] Spider silk fragments

[0539] Spider silk is a natural polymer composed of three domains: a repetitive central core domain that dominates the protein chain and non-repetitive N- and C-terminal domains. The large core domain is organized in a block copolymer-like arrangement, with two basic sequences—a crystalline polypeptide (poly(A) or poly(GA)) and a less crystalline polypeptide (GGX or GPGXX)—alternating within the core domain. Dragline silk is a protein complex composed of major ampullate dragline protein 1 (MaSp1) and major ampullate dragline protein 2 (MaSp2). Both silks are approximately 3,500 amino acids long. MaSp1 is found in the fiber core and periphery, while MaSp2 forms clusters in certain core regions. The large central domains of MaSp1 and MaSp2 are organized in a block copolymer-like arrangement, with two basic sequences—a crystalline polypeptide (poly(A) or poly(GA)) and a less crystalline polypeptide (GGX or GPGXX)—alternating within the core domain. Specific secondary structures have been assigned to poly(A) / (GA), GGX, and GPGXX motifs, comprising β-sheets, α-helices, and β-helices, respectively. The primary sequence, composition, and secondary structural elements of the repetitive core domain determine the mechanical properties of spider silk, while the non-repetitive N- and C-terminal domains are crucial for storing the liquid silk dope in the lumen and forming fibers in the spinning duct.

[0540] The main difference between MaSp1 and MaSp2 is the presence of proline (P) residues, which make up 15% of the total amino acid content in MaSp2, while MaSp1 contains no proline. By counting the number of proline residues in dragline silk from the spider N. clavipes, it was possible to estimate the presence of both proteins in the fiber: 81% MaSp1 and 19% MaSp2. Different spiders have different ratios of MaSp1 and MaSp2. For example, dragline silk fibers from the orb weaver Argiope aurantia contain 41% MaSp1 and 59% MaSp2. This variation in the ratio of large ampullate silk can determine the properties of the silk fiber.

[0541] At least seven different types of silk proteins are known for a single species of Theridiidae spider. Silks differ in their primary sequence, physical properties, and function. For example, dragline silk, used to construct the framework, radii, and lifelines of spiders, is known for its exceptional mechanical properties, including strength, toughness, and elasticity. Weight for weight, spider silk is tougher than steel and Kevlar.

[0542] Flageliform silk, found in capture spirals, has an extensibility of up to 500%. Small ampullate silk, found in auxiliary spirals of orb-webs and prey wrapping, has a high tenacity and strength similar to large ampullate silk, but does not supercontract in water.

[0543] Spider silks are known for their high tensile strength and toughness. Recombinant silk proteins also impart advantageous properties to cosmetic or dermatological compositions, particularly improved hydration or softening, good film-forming properties, and low surface density. The diverse and unique biomechanical properties, combined with biocompatibility and slow degradation rates, make spider silk an excellent candidate as a biomaterial for tissue engineering, guided tissue repair, and drug delivery, in cosmetic products (e.g., nail and hair strengtheners, skin care products), and in industrial materials (e.g., nanowires, nanofibers, surface coatings).

[0544] In one embodiment, the silk protein may include a polypeptide derived from a natural spider silk protein. The polypeptide is not particularly limited as long as it is derived from a natural spider silk protein, and examples of the polypeptide include natural spider silk proteins and recombinant spider silk proteins, such as variants, analogs, derivatives, etc. of natural spider silk proteins. In terms of excellent toughness, the polypeptide may be derived from the main dragline silk protein produced in the spider's ampulla. Examples of the main dragline silk protein include the major ampulla silk proteins MaSp1 and MaSp2 from the spider's genus Nephilaclavipes and ADF3 and ADF4 from the spider Araneus diadematus. Examples of polypeptides derived from the main dragline silk protein include variants, analogs, derivatives, etc. of the main dragline silk protein. In addition, the polypeptide may be derived from the whip gland silk protein produced in the spider's whip gland. Examples of whip gland silk proteins include whip gland silk proteins derived from the spider's genus Nephilaclavipes, etc.

[0545] Examples of polypeptides derived from the major dragline protein include polypeptides containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2 (1), preferably polypeptides containing five or more units thereof, and more preferably polypeptides containing ten or more units thereof. Alternatively, the polypeptide derived from the major dragline protein may be a polypeptide containing units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) and having at the C-terminus an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 of U.S. Patent No. 9,051,453 (which is incorporated herein by reference in its entirety), or an amino acid sequence having 90% or more homology to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 of U.S. Patent No. 9,051,453 (which is incorporated herein by reference in its entirety). In the polypeptide derived from the major dragline protein, the units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) may be the same as or different from each other. In the case of producing a recombinant protein using a microorganism such as Escherichia coli as a host, the molecular weight of the polypeptide derived from the major dragline protein is 500 kDa or less, or 300 kDa or less, or 200 kDa or less in consideration of productivity.

[0546] In formula (1), REP1 refers to polyalanine. In REP1, the number of consecutively arranged alanine residues is preferably 2 or more, more preferably 3 or more, further preferably 4 or more, and particularly preferably 5 or more. In addition, in REP1, the number of consecutively arranged alanine residues is preferably 20 or less, more preferably 16 or less, further preferably 12 or less, and particularly preferably 10 or less. In formula (1), REP2 is an amino acid sequence consisting of 10 to 200 amino acid residues. The total number of glycine, serine, glutamine and alanine residues contained in the amino acid sequence is 40% or more, preferably 60% or more, and more preferably 70% or more relative to the total number of amino acid residues contained therein.

[0547] In the primary dragline, REP1 corresponds to the crystalline region of the fiber, where crystalline beta sheets form, and REP2 corresponds to the amorphous region of the fiber, where most of the strands lack a regular structure and are more flexible. Furthermore, [REP1-REP2] corresponds to a repeating region (repeating sequence) consisting of crystalline and amorphous regions, which is a characteristic sequence of dragline proteins.

[0548] Recombinant silk fragment

[0549] In some embodiments, the recombinant silk protein refers to a recombinant spider silk polypeptide, a recombinant insect silk polypeptide or a recombinant spidroin polypeptide. In some embodiments, the recombinant silk protein fragments disclosed herein include recombinant spider silk polypeptides of the family Araneidae or Araneoids, or recombinant insect silk polypeptides of the silkworm (Bombyx mori). In some embodiments, the recombinant silk protein fragments disclosed herein include recombinant spider silk polypeptides of the family Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein include block copolymers having repeating units derived from natural spider silk polypeptides of the family Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein include block copolymers having synthetic repeating units derived from spider silk polypeptides of the family Araneidae or Araneoids and block copolymers of non-repeating units derived from natural repeating units of spider silk polypeptides of the family Araneidae or Araneoids.

[0550] Recent advances in genetic engineering have provided routes for producing various types of recombinant silk proteins. Recombinant DNA technology has been used to provide a more practical source of silk proteins. As used herein, "recombinant silk protein" refers to a synthetic protein produced heterologously in a prokaryotic or eukaryotic expression system using genetic engineering methods.

[0551] Various methods for synthesizing recombinant silk peptides are known and described by Ausubel et al., Current Protocols in Molecular Biology §8 (John Wiley & Sons 1987, (1990)), which is incorporated herein by reference. The Gram-negative, rod-shaped bacterium Escherichia coli (E. coli) is a recognized host for industrial-scale protein production. Therefore, most recombinant silk has been produced in E. coli. E. coli is easy to manipulate, has a short generation time, is relatively low-cost, and can be scaled up for larger protein production.

[0552] Recombinant silk proteins can be produced by transforming eukaryotic or prokaryotic systems containing cDNA encoding for silk proteins, fragments of such proteins, or analogs of such proteins. The recombinant DNA approach is capable of producing recombinant silk with programmed sequence, secondary structure, architecture, and precise molecular weight. There are four main steps in this method: (i) designing and assembling the synthetic silk-like gene into a gene "cassette," (ii) inserting this fragment into a recombinant DNA vector, (iii) transforming this recombinant DNA molecule into a host cell, and (iv) expression and purification of the selected clone.

[0553] The term "recombinant vector" as used herein includes any vector known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). Said vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the desired coding sequence and the appropriate DNA sequences necessary for expressing the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast or plant) or in an in vitro expression system. Cloning vectors are generally used for engineering and amplification of specific desired DNA fragments and may lack the functional sequences required for expressing the desired DNA fragments.

[0554] The prokaryotic system includes gram-negative bacteria or gram-positive bacteria. The prokaryotic expression vector may include a host organism recognizable origin of replication, a functional homologous or heterologous promoter in the host, a spidroin, a fragment of this protein, or a DNA sequence encoding a similar protein. Non-limiting examples of prokaryotic expression organisms are Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, Anabaena, Caulobacter, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus (such as Bacillus subtilis), Brevibacterium, Corynebacterium, Rhizobium (Sinorrhiza), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylbacterium, Propionibacterium, Staphylococcus or Streptomyces cells.

[0555] Eukaryotic systems include yeast and insect, mammalian or plant cells. In this case, the expression vector may include a yeast plasmid origin of replication or an autonomously replicating sequence, a promoter, a DNA sequence encoding a spider silk protein, a fragment or a similar protein, a polyadenylation sequence, a transcription termination site and finally, a selector gene. Non-limiting examples of eukaryotic expression organisms include yeasts such as Saccharomyces cerevisiae, Pichia pastoris, basidiosporogenous yeasts, ascosporogenous yeasts, filamentous fungi such as Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, Acremonium chrysogenum, Candida, Hansenula, Kluyveromyces, Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces, Pichia pastoris. (e.g., Pichia pastoris) or Yarrowia cells, mammalian cells, such as HeLa cells, COS cells, CHO cells, etc., insect cells, such as Sf9 cells, MEL cells, etc., "insect host cells", such as Spodoptera frugiperda or Trichoplusia ni cells, SF9 cells, SF-21 cells or High-Five cells, wherein SF-9 and SF-21 are ovary cells from Spodoptera frugiperda, and High-Five cells are egg cells from Trichoplusia ni, "plant host cells", such as tobacco, potato or pea cells.

[0556] A variety of heterologous host systems have been developed for the production of different types of recombinant silk. Recombinant partial spider silk proteins and engineered silk have been cloned and expressed in bacteria (Escherichia coli), yeast (Pichia pastoris), insects (Bombyx mori larvae), plants (tobacco, soybean, potato, Arabidopsis), mammalian cell lines (BHT / hamster), and transgenic animals (mouse, goat). Most silk proteins produced have an N-terminal or C-terminal His-tag to facilitate purification and produce sufficient protein.

[0557] In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems may include transgenic animals and plants. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include bacteria, yeast, and mammalian cell lines. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include Escherichia coli. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include transgenic silkworms (B. mori) generated using genome editing technology (e.g., CRISPR).

[0558] The recombinant silk proteins in the present disclosure comprise synthetic proteins based on the repeating units of natural silk proteins. In addition to the synthetic repeating silk protein sequences, these may further comprise one or more natural non-repeating silk protein sequences.

[0559] In some embodiments, "recombinant silk protein" refers to recombinant silk protein or fragments thereof. Recombinant production of fibroin and sericin has been reported. Various hosts have been used for such production, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas, Rhodopseudomonas, Bacillus, and Streptomyces. See EP 0230702, which is incorporated herein by reference in its entirety.

[0560] Also provided herein is the design and biosynthesis of fibroin-like multiblock polymers comprising the GAGAGX hexapeptide (X is A, Y, V, or S) derived from the repeating domain of the Bombyx mori silk heavy chain (H chain).

[0561] In some embodiments, the present disclosure provides a silk protein-like multi-block polymer derived from a repeating domain of a Bombyx mori silk heavy chain (H chain) comprising a GAGAGS hexapeptide repeating unit. The GAGAGS hexapeptide is the core unit of the H chain and plays an important role in the formation of the crystalline domain. The silk protein-like multi-block polymer containing the GAGAGS hexapeptide repeating unit spontaneously aggregates into a β-pleated structure similar to natural silk core protein, wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0562] In some embodiments, the present disclosure provides a silk peptide-like multi-block copolymer composed of a GAGAGS hexapeptide repeating fragment derived from the H chain of a Bombyx mori silk heavy chain and a mammalian elastin VPGVG motif produced by Escherichia coli. In some embodiments, the present disclosure provides a fusion silk fibroin composed of a GAGAGS hexapeptide repeating fragment derived from the H chain of a Bombyx mori silk heavy chain and GVGVP produced by Escherichia coli, wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0563] In some embodiments, the present disclosure provides a mixture consisting of (GAGAGS) 16 In some embodiments, the present disclosure provides a recombinant silkworm protein consisting of (GAGAGS) 16 Repeated fragments and non-repeated fragments produced by E. coli (GAGAGS) 16 -F-COOH, (GAGAGS) 16 -FF-COOH, (GAGAGS) 16 -FFF-COOH, (GAGAGS) 16 -FFFF-COOH, (GAGAGS) 16 -FFFFFFFF-COOH, (GAGAGS) 16-FFFFFFFFFFFF-COOH, wherein F has the following amino acid sequence SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG, and wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0564] In some embodiments, "recombinant silk protein" refers to a recombinant spider silk protein or a fragment thereof. Production of recombinant spider silk proteins based on partial cDNA cloning has been reported. The recombinant spider silk protein thus produced comprises a portion of a repeating sequence derived from the spider rod thread spidroin 1 protein from the spider Nephila spidroin. See Xu et al. (Proc. Natl. Acad. Sci. USA, 87: 7120-7124 (1990). The cDNA cloning and recombinant synthesis of a portion of the repeat sequence encoding Spidroin 2, the second fibroin of the dragline silk from the spider Nephila clavipes is described in J. Biol. Chem., 1992, Vol. 267, pp. 19320-19324. The recombinant synthesis of spider silk proteins comprising protein fragments and variants by transforming E. coli is described in U.S. Patents 5,728,810 and 5,989,894. The cDNA cloning and expression of the ampullate gland spider silk protein are described in U.S. Patents 5,733,771 and 5,756,677. The cDNA cloning of the whip gland silk protein of the spinning spider is described in U.S. Patent 5,994,099. U.S. Patent 6,268,169 describes the recombinant synthesis of a spider silk-like protein derived from a repetitive peptide sequence present in the natural spider dragline silk of the genus Nephila by recombinant expression systems in Escherichia coli, Bacillus subtilis and Pichia pastoris. WO 03 / 020916 describes a spider silk-like protein having large ampullate glands derived from the golden orb spiders Nephila madagascariensis, Nephila senegalensis, Tetragnatha kauaiensis, Tetragnatha versicolor, Argiope aurantia, Argiope trifasciata, Gasteracanthamammosa and Latrodectus geometricus, the whip glands of Argiope trifasciata, the ampullate glands of Dolomedes tenebrosus, two sets of silk glands of Plectreurys tristis and the mygalomorph Euagrus cDNA cloning and recombinant production of a spider silk protein encoding a repetitive sequence from the silk gland of E. chisoseus. Each of the above references is incorporated herein by reference in its entirety.

[0565] In some embodiments, the recombinant spidroin protein is a hybrid protein of a spidroin protein and an insect silk protein, a spidroin protein and collagen, a spidroin protein and elastic protein, or a spidroin protein and keratin. The spider silk repeating unit comprises or consists of the amino acid sequence of a region comprising or consisting of at least one peptide motif that is repeated in a naturally occurring large ampullate polypeptide, such as a dragline silk polypeptide, a small ampullate polypeptide, a whip-like polypeptide, an aggregated silk polypeptide, a grape-like silk polypeptide, or a pyriform silk polypeptide.

[0566] In some embodiments, the recombinant spidroin protein of the present disclosure comprises a synthetic spidroin protein comprising repeating units derived from a natural spidroin protein, a consensus sequence, and optionally one or more natural non-repetitive spidroin protein sequences. The repeating units of the natural spidroin polypeptide may comprise a dragline silk polypeptide or a whip gland spidroin polypeptide of the family Araneidae or Araneoids.

[0567] As used herein, a spider silk "repeat unit" comprises or consists of at least one peptide motif that is repeated in a naturally occurring macroampullar gland polypeptide, such as a dragline silk polypeptide, a microampullar gland polypeptide, a whip gland polypeptide, a polymorphic gland silk polypeptide, a grape gland silk polypeptide, or a piriform gland silk polypeptide. A "repeat unit" refers to a region of amino acid sequence that corresponds to or consists of at least one peptide motif (e.g., AAAAAA or GPGQQ) that is repeated in a naturally occurring silk polypeptide (e.g., MaSpI, ADF-3, ADF-4, or Flag) (i.e., the same amino acid sequence) or a region that corresponds to an amino acid sequence that is substantially similar thereto (i.e., a variant amino acid sequence). A "repeat unit" having an amino acid sequence that is "substantially similar" to the corresponding amino acid sequence in a naturally occurring silk polypeptide (i.e., a wild-type repeat unit) is also similar in its properties, for example, a silk protein comprising a "substantially similar repeat unit" remains insoluble and maintains its insolubility. A "repeat unit" having an amino acid sequence that is "identical" to the amino acid sequence of a naturally occurring silk polypeptide can, for example, be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI, MaSpII, ADF-3 and / or ADF-4. A "repeat unit" having an amino acid sequence that is "substantially similar" to the amino acid sequence of a naturally occurring silk polypeptide can, for example, be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI, MaSpII, ADF-3 and / or ADF-4 but having one or more amino acid substitutions at specific amino acid positions.

[0568] As used herein, the term "consensus peptide sequence" refers to an amino acid sequence containing an amino acid that frequently occurs at a certain position (e.g., "G") and in which other amino acids that are not further identified are replaced by the placeholder "X." In some embodiments, the consensus sequence is at least one of the following: (i) GPGXX, wherein X is an amino acid selected from A, S, G, Y, P, and Q; (ii) GGX, wherein X is an amino acid selected from Y, P, R, S, A, T, N, and Q, preferably Y, P, and Q; (iii) A x , where x is an integer from 5 to 10.

[0569] The consensus peptide sequence of GPGXX and GGX, i.e. the glycine-rich motif, provides flexibility to silk polypeptides and therefore provides flexibility to the threads formed by the silk proteins containing the motif. In detail, the iterative GPGXX motif forms a rotating helical structure, which gives the silk polypeptide elasticity. Both the large ampullate and whip-shaped gland silks have the GPGXX motif. The iterative GGX motif is associated with a helical structure with three amino acids per turn and is present in most spider silks. The GGX motif can provide additional elasticity to the silk. The iterative polyalanine Ax (peptide) motif forms a crystalline β-pleated structure to provide strength to the silk polypeptide, as described, for example, in WO 03 / 057727.

[0570] In some embodiments, the recombinant spider silk protein of the present disclosure comprises two identical repeating units, each of which comprises at least one, preferably one, amino acid sequence selected from the group consisting of GGRPSDTYG and GGRPSSSYG derived from resilin, an elastomeric protein found in most arthropods that provides low stiffness and high strength.

[0571] As used herein, "non-repeating unit" refers to an amino acid sequence that is "substantially similar" to the corresponding non-repeating (carboxyl-terminal) amino acid sequence in naturally occurring dragline silk polypeptides (i.e., wild-type non-repeating (carboxyl-terminal) units), preferably ADF-3, ADF-4, NR3, NR4 of the spider Araneus cruciferus (also described in U.S. Patent No. 9,217,017, which is incorporated herein by reference in its entirety), the C16 peptide comprising 16 repeats of the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (spidroin protein eADF4, molecular weight 47.7 kDa, AMSilk), or the amino acid sequence modified from the native sequence of ADF4 from Araneus cruciferus. Non-repeating ADF-4 and its variants exhibit efficient assembly properties.

[0572] Among synthetic spider silk proteins, the recombinant silk proteins of the present disclosure, in some embodiments, comprise a C16 protein having the polypeptide sequence SEQ ID NO: 1 (as described in U.S. Patent No. 8,288,512, which is incorporated herein by reference in its entirety). In addition to the polypeptide sequence shown in SEQ ID NO: 1 (as described in U.S. Patent No. 8,288,512), functional equivalents, functional derivatives, and salts of this sequence are also specifically included.

[0573] As used herein, "functional equivalents" refer to mutants which have an amino acid different from the specifically mentioned amino acid in at least one sequence position of the above-mentioned amino acid sequences.

[0574] In some embodiments, the recombinant spidroin proteins of the present disclosure comprise an effective amount of at least one natural or recombinant silk protein, including spidroin proteins corresponding to Spidroin major 1 described by Xu et al., PNAS, USA, 87, 7120, (1990), Spidroin major 2 described by Hinman and Lewis, J. Biol. Chem., 267, 19320, (1922), recombinant spidroin proteins as described in U.S. patent application 2016 / 0222174 and U.S. Patents 9,051,453, 9,617,315, 9,689,089, 8,173,772, 8,642,734, 8,367,8038,097,583, 8,030,024, 7,754,851, 7,148,039, 7,060,260, or patent application WO 95 / 25165. Each of the above-cited references is incorporated herein by reference in its entirety. Additional recombinant spidroins suitable for use in the recombinant RSPF of the present disclosure include ADF3 and ADF4 from the "major ampulla gland" of the spider Araneus crusae.

[0575] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US 2004590196, US 7,754,851, US 2007654470, US 7,951,908, US 2010785960, US 8,034,897, US 20090263430, US 2008226854, US 20090123967, US 2005712095, US 2007991037, US 20090162896, US 200885266, US 8,372,436, US 2007989907, US 2009267596, US 2010319542, US 2009265344, US 2012684607, US 2004583227, US 8,030,024, US2006643569, US 7,868,146, US 2007991916, US 8,097,583, US 2006643200, US 8,729,238, US 8,877,903, US 20190062557, US 20160280960, US 20110201783, US 2008991916, US 2011986662, US 2012697729, US 20150328363, US 9,034,816、US 20130172478、US 9,217,017, US 20170202995, US 8,721,991, US 2008227498, US 9,233,067, US 8,288,512, US 2008161364, US 7,148,039, US1999247806, US 2001861597, US 2004887100, US 9,481,719, US 8,765,688, US 200880705, US 2010809102, US 8,367,803, US 2010664902, US 7,569,660, US 1999138833、US 2000591632, US 20120065126, US 20100278882, US2008161352, US 20100015070, US 2009513709, US 20090194317, US 2004559286, US200589551, US 2008187824, US 20050266242, US 20050227322 and US 20044418.

[0576] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US 20190062557, US 20150284565, US 201...

Claims

1. A peptide or protein fragment comprising a plurality of amino acids selected from the group consisting of M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P and W, wherein at least one of the amino acids is modified, substituted or replaced. 2 . The peptide or protein fragment according to claim 1 , wherein the peptide or protein fragment is a fibroin peptide or protein fragment comprising an amino acid modification, substitution or replacement as defined in claim 1 . 3 . The peptide or protein fragment according to claim 2 , wherein the fibroin is a fibroin heavy chain, a fibroin light chain or a fibroin hexamer.

4. The peptide or protein fragment according to any one of claims 1 to 3, wherein the peptide or protein fragment comprises about 2 to about 100 amino acids.

5. The peptide or protein fragment according to any one of claims 1 to 4, wherein the peptide or protein fragment comprises one to five modifications, substitutions and / or replacements.

6. The peptide or protein fragment according to any one of claims 2 to 5, wherein the fibroin is a fibroin heavy chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the fibroin heavy chain.

7. The peptide or protein fragment according to claim 6, wherein the modification, substitution and / or replacement is selected from the group consisting of asparagine to aspartic acid modification, substitution and / or replacement, glutamine to glutamic acid modification, substitution and / or replacement, and methionine to methionine oxide modification, substitution and / or replacement.

8. The peptide or protein fragment according to claim 6 or 7, wherein the modification, substitution and / or replacement is at Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262.

9. The peptide or protein fragment according to any one of claims 2 to 5, wherein the fibroin is a fibroin light chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain.

10. The peptide or protein fragment according to claim 9, wherein the modification, substitution and / or replacement is selected from the group consisting of asparagine to aspartic acid modification, substitution and / or replacement, glutamine to glutamic acid modification, substitution and / or replacement, and methionine to methionine oxide modification, substitution and / or replacement.

11. The peptide or protein fragment according to claim 9 or 10, wherein the modification, substitution and / or replacement is at N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255.

12. A peptide or protein fragment according to any one of claims 2 to 5, wherein the fibroin is a fibroin hexamer (p25), and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 220 of the fibroin hexamer (p25).

13. The peptide or protein fragment according to claim 9, wherein the modification, substitution and / or replacement is selected from the group consisting of asparagine to aspartic acid modification, substitution and / or replacement, glutamine to glutamic acid modification, substitution and / or replacement, and methionine to methionine oxide modification, substitution and / or replacement.

14. The peptide or protein fragment according to claim 12 or 13, wherein the modification, substitution and / or replacement is at Q62, N93, M120, N149, N172, N174 and / or N202.

15. A preparation comprising the peptide or protein fragment according to any one of claims 1 to 14.

16. A composition comprising a plurality of peptides or protein fragments, each peptide or protein fragment comprising a plurality of amino acids selected from the group consisting of M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, S, H, P and W, wherein at least one of the amino acids is modified, substituted or replaced. 17 . The composition according to claim 16 , wherein the plurality of peptides or protein fragments comprise a fibroin peptide or protein fragment comprising an amino acid modification, substitution or replacement as defined in claim 16 . The composition according to claim 17 , wherein the silk fibroin is a silk fibroin heavy chain, a silk fibroin light chain or a silk fibroin hexamer.

19. The composition of any one of claims 16 to 18, wherein the peptide or protein fragment comprises from about 2 to about 100 amino acids.

20. The composition of any one of claims 1 to 4, wherein the peptide or protein fragment comprises one to five modifications, substitutions and / or replacements.

21. The composition according to any one of claims 17 to 20, wherein the silk fibroin is a silk fibroin heavy chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the silk fibroin heavy chain.

22. The composition of claim 21, wherein the modification, substitution and / or replacement is selected from the group consisting of asparagine to aspartic acid modification, substitution and / or replacement, glutamine to glutamic acid modification, substitution and / or replacement, and methionine to methionine oxide modification, substitution and / or replacement.

23. The composition of claim 21 or 22, wherein the modification, substitution and / or replacement is at Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262.

24. The composition according to any one of claims 17 to 20, wherein the fibroin is a fibroin light chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain.

25. The composition of claim 24, wherein the modification, substitution and / or replacement is selected from the group consisting of an asparagine to aspartic acid modification, substitution and / or replacement, a glutamine to glutamic acid modification, substitution and / or replacement, and a methionine to methionine oxide modification, substitution and / or replacement.

26. The composition of claim 24 or 25, wherein the modification, substitution and / or replacement is at N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255.

27. A composition according to any one of claims 17 to 20, wherein the silk fibroin is a silk fibroin hexamer (p25) chain, and wherein the modification, substitution and / or replacement is located at a position corresponding to any one of positions 1 to 220 of the silk fibroin hexamer (p25) chain.

28. The composition of claim 27, wherein the modification, substitution and / or replacement is selected from the group consisting of asparagine to aspartic acid modification, substitution and / or replacement, glutamine to glutamic acid modification, substitution and / or replacement, and methionine to methionine oxide modification, substitution and / or replacement.

29. The composition of claim 27 or 28, wherein the modification, substitution and / or replacement is at Q62, N93, M120, N149, N172, N174 and / or N202.

30. The composition of any one of claims 17 to 29, wherein each modification, substitution and / or replacement independently ranges from about 1% to about 99% in the composition.

31. A composition according to claim 32, wherein the modification %, substitution % and / or replacement % is defined as (the number of peptides or protein fragments comprising a modification, substitution and / or replacement at a particular position divided by the total number of peptides or protein fragments comprising the particular position, whether or not comprising a modification, substitution and / or replacement) x 100.

32. An article comprising the composition of any one of claims 17 to 31.

33. A composition comprising multiple peptides or protein fragments of fibroin heavy chain, fibroin light chain and / or fibroin hexamer (p25), wherein the composition comprises one or more fractions, wherein the multiple peptides or protein fragments comprise fibroin peptides or protein fragments containing amino acid modifications, substitutions or replacements.

34. The composition of claim 33, wherein the plurality of peptide or protein fragments have a weight average molecular weight (MW) selected from the group consisting of about 1 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, or about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 250 kDa. w ) and a polydispersity of 1 to about 1.

7.

35. The composition of claim 33, wherein the plurality of peptide or protein fragments have a weight average molecular weight (MW) selected from the group consisting of about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, or about 160 kDa to about 180 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

36. The composition of claim 33, wherein the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (MW) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

37. The composition of claim 33, wherein the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (MW) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 120 kDa. w ) and a polydispersity of 1 to about 1.

1.

38. The composition of claim 33, wherein the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (MW) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

39. The composition of claim 33, wherein the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (MW) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ) and a polydispersity of 1 to about 1.

1.

40. The composition of claim 33, wherein the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (MW) selected from about 20 kDa to about 40 kDa or about 40 kDa to about 60 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

41. The composition of claim 33, wherein the one or more fractions are selected from AS77, AS78, AS79, AS80, and AS81.

42. The composition of claim 33, wherein the one or more fractions are selected from AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89.

43. The composition of claim 33, wherein the one or more fractions are selected from AS90, AS91, AS92, AS93, and AS94.

44. The composition of claim 33, wherein the one or more fractions are selected from AS95, AS96, AS97, AS98, AS99, and AS100.

45. The composition of claim 33, wherein the plurality of peptide or protein fragments have a weight average molecular weight (MW) selected from the group consisting of about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 220 kDa. w ) and a polydispersity of 1 to about 1.

7.

46. The composition of claim 33, wherein the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (MW) selected from the group consisting of about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w ) and a polydispersity of 1 to about 1.2 or 1 to about 1.

3.

47. The composition of claim 33, wherein the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (MW) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

48. The composition of claim 33, wherein the plurality of peptides or protein fragments in the fraction have a weight average molecular weight (MW) selected from the group consisting of about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w ) and a polydispersity of 1 to about 1.2 or 1 to about 1.

3.

49. The composition of claim 33, wherein the one or more fractions are selected from AS101, AS102, AS103, AS104, and AS105.

50. The composition of claim 33, wherein the one or more fractions are selected from AS106, AS107, AS108, AS109, AS110, and AS111.

51. The composition of any one of claims 33 to 50, wherein amino acids are selected from the group consisting of M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W.

52. The composition of any one of claims 33 to 51, wherein the peptide or protein fragment comprises from about 2 to about 100 amino acids.

53. A composition according to any one of claims 33 to 52, wherein the peptide or protein fragment comprises one to five modifications, substitutions and / or replacements.

54. A composition according to any one of claims 33 to 53, wherein the silk fibroin is a silk fibroin heavy chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the silk fibroin heavy chain.

55. A composition according to any one of claims 33 to 53, wherein the fibroin is a fibroin light chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain.

56. A composition according to any one of claims 33 to 53, wherein the silk fibroin is a silk fibroin hexamer (p25) chain, and wherein the modification, substitution and / or replacement is located at a position corresponding to any position from 1 to 220 of the silk fibroin hexamer (p25) chain.

57. according to the composition described in any one of claim 33 to 56, wherein modification, substitution and / or replacement is selected from asparagine to aspartic acid modification, substitution and / or replacement, glutamine to glutamic acid modification, substitution and / or replacement, and methionine to methionine oxide modification, substitution and / or replacement.

58. according to the composition described in any one of claim 33 to 53, wherein modification, substitution and / or replacement is located at the silk fibroin heavy chain position selected from Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262.

59. according to the composition described in any one of claim 33 to 53, wherein modification, substitution and / or replacement are positioned at the silk fibroin light chain position selected from N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255.

60. A composition according to any one of claims 33 to 53, wherein the modification, substitution and / or replacement is located at a position of the fibroin hexamer (p25) selected from Q62, N93, M120, N149, N172, N174 and / or N202.

61. The composition of any one of claims 33 to 60, wherein each modification, substitution and / or replacement independently ranges from about 1% to about 99% in the composition.

62. according to the compositions described in any one in claim 33 to 61, wherein modification %, substitution % and / or replacement % are defined as (the number of peptides or protein fragments comprising modifications, substitutions and / or replacements at a specific position divided by the total number of peptides or protein fragments comprising said specific position, no matter whether comprising modifications, substitutions and / or replacements) × 100.

63. The composition of any one of claims 33 to 62, wherein the molecular weight is determined by MALS.

64. An article comprising the composition of any one of claims 33 to 63.

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