Elastomer protein

Abstract

The present invention relates to elastomeric proteins and elastomeric protein production. In particular, the invention is directed to elastomeric protein sequences and includes methods and compositions for the production of elastomeric protein sequences, such as expression constructs, and host cells, and also includes compositions produced from the elastomeric protein sequences.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 446,230, filed January 13, 2017, which is incorporated herein by reference in its entirety for all purposes.

[0002] Technical field This disclosure generally relates to elastomer proteins and elastomer protein production. Specifically, this disclosure relates to elastomer protein sequences, expression constructs, host cells, and solids. [Background technology]

[0003] background Elastomer proteins are polypeptides that exhibit viscoelastic mechanical properties and include elastin, resilin, abductin, and octopus artery elastomers. Resilin is a particularly interesting elastomer protein because it dissipates very little energy during loading and unloading. Resilin is found in many insects, and its low energy dissipation enables the extraordinary ability of many insect species to jump or flap their wings with extreme efficiency. Due to its unique properties, resilin is an interesting elastomer material that could have many industrial applications. However, resilin exists only in very small amounts in nature and therefore cannot be obtained cost-effectively through insect rearing.

[0004] Variants of natural resilin and resilin-like proteins (based on resilin sequences) have been recombinantly produced in E. coli (E. coli) cultures by numerous groups. The recombinant proteins are isolated by lysing the cells to extract them and using affinity chromatography techniques for purification (Elvin et al., 2005; Charati et al., 2009; McGann et al., 2013). Recombinant resilin and resilin-like proteins are crosslinked targeting tyrosine residues that also form crosslinks in natural resilin (see, e.g., Elvin et al., 2005; Qin et al., 2011). Recombinant resilin can also be crosslinked targeting lysine residues (Li et al., 2011) or cysteine ​​residues (McGann et al., 2013). Cross-linked recombinant resilin and resilin-like proteins exhibited mechanical properties similar to natural resilin and possessed elastic energy values ​​higher than 90% (Elvin et al., 2005; Qin et al., 2011; Li et al., 2011).

[0005] In one study, 70–80 mg of recombinant resilin-like protein was produced per liter of E. coli culture, and the resilin-like protein was purified by Ni-NTA affinity chromatography (Charati et al., 2009). A more efficient expression system was developed, which produced 300–450 mg / L of recombinant resilin-like protein from E. coli host cells (Lyons, et al., 2009). A more efficient method for purifying resilin-like protein from lysed E. coli host cells based on heating after salt precipitation has also been developed (Qin et al., 2011; Lyons et al., 2009). However, an improved system for expressing and purifying elastomer proteins (e.g., resilin and resilin-like proteins) with higher production volumes is desired to provide larger-scale and more efficient protein production.

[0006] One drawback of recovering expressed proteins by simple precipitation-based purification techniques after cell lysis is that the resulting proteins tend to have low purity due to contamination of the target protein with cellular proteins derived from the lysed cells. Low purity can result in a variety of product defects, including low elastic energy. Furthermore, intracellular accumulation of proteins can lead to toxicity and thus a decrease in the efficiency of recombinant elastomer protein production. Therefore, there is a need for improved methods for the expression and purification of recombinant elastomer proteins, including methods for recovering elastomer proteins from extracellular components. There is also a need for improved methods for the expression and purification of recombinant elastomer proteins (e.g., resilins and resilin-like proteins) with greater production efficiency. [Overview of the project]

[0007] According to several embodiments, methods for preparing compositions comprising recombinant resilin proteins are provided herein, comprising the steps of: culturing a population of recombinant host cells in a ferment, wherein the recombinant host cells comprise a vector comprising a secretory resilin coding sequence, and the recombinant host cells secrete recombinant resilin proteins encoded by the secretory resilin coding sequence; and purifying recombinant resilin proteins from the ferment.

[0008] In some embodiments, recombinant resilin proteins are full-length or truncated native resilin proteins. In some forms, native resilience is found in the following insects: Drosophila sechellia, Acromyrmex echinatior, Aeshna, Haematobia irritans, Ctenocephalides felis, Bombus terrestris, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Pediculus humanus corporis, Anopheles gambiae, Glossina morsitans, Atta cephalotes, and Anopheles darlingji. The recombinant resilin protein is derived from organisms selected from the group consisting of *Drosophila darlingi*, *Acyrthosiphon pisum*, *Drosophila virilis*, *Drosophila erecta*, *Lutzomyia longipalpis*, *Rhodnius prolixus*, *Solenopsis invicta*, *Culex quinquefasciatus*, *Bactrocera cucurbitae*, and *Trichogramma pretiosum*. In some embodiments, the recombinant resilin protein contains SEQ ID NO:1. In some embodiments, the recombinant resilin protein contains SEQ ID NO:4.

[0009] In some embodiments, the recombinant resilin protein contains an α-junction factor secretion signal. In some embodiments, the recombinant resilin protein contains a FLAG tag. In some embodiments, the vector contains multiple secreted resilin coding sequences.

[0010] In some embodiments, the recombinant host cell is a yeast cell. In some embodiments, the yeast cell is a methanolic yeast cell. In some embodiments, the recombinant host cell is a species selected from the group consisting of Pichia (Komagataella) pastoris, Hansenula polymorpha, Arxula adeninivorans, Yarrowia lipolytica, Pichia (Scheffersomyces) stipitis, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis.

[0011] In some embodiments, recombinant host cells produce recombinant resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour. In some embodiments, recombinant host cells produce a secreted fraction of recombinant resilin, which is more than 50% of the total recombinant resilin protein expressed by the recombinant host cells. In some embodiments, recombinant host cells secrete recombinant resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour. In some embodiments, more than 80% of the recombinant resilin is present outside the recombinant host cells in the ferment. In some embodiments, the ferment contains at least 2 g of recombinant resilin per liter.

[0012] In some embodiments, the step of purifying recombinant resilin protein includes centrifugation of the ferment to produce a first pellet fraction and a first supernatant fraction; and isolation of recombinant resilin protein from the first pellet fraction. In some embodiments, the step of purifying recombinant resilin protein further includes adding chaotrope to the first pellet fraction to produce a solution in which recombinant resilin protein is soluble; centrifugation of the first pellet fraction containing chaotrope to produce a second supernatant fraction and a second pellet fraction; and isolation of soluble full-length resilin from the second supernatant fraction.

[0013] In some embodiments, vectors comprising secreted resilin coding sequences are provided herein. In some embodiments, the secreted resilin coding sequence encodes a full-length or truncated native resilin. In some embodiments, the secreted resilin coding sequence encodes a modified full-length or truncated native resilin. In some embodiments, the modified resilin comprises the addition, deletion, substitution, or repositioning of an amino acid residue, wherein the amino acid residue can be crosslinked with another resilin.

[0014] In some embodiments, the full-length or shortened native resilin is derived from organisms selected from the group consisting of Drosophila melanogaster, Drosophila spp., Aeshna fuscipes, Stingray flies, Cat fleas, Bombus ignitus, Confused flour beetles, Honeybees, Parasitic wasps, Body lice, Anopheles gambia, Glossina moorcitans, Atta cephalotes, Anopheles darlingzi, Pea aphids, Drosophila melanogaster, Drosophila kirishimaensis, Sand flies, Assassin bugs, Red imported fire ants, Culex pipiens, Culex tritaeniorhynchus, Trichogramma plethiosum.

[0015] In some embodiments, the secreted resilin coding sequence encodes a polypeptide comprising SEQ ID NO:1. In some embodiments, the secreted resilin coding sequence encodes a polypeptide comprising SEQ ID NO:4. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising one or more A repeats or pseudo-A repeats. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising one or more B repeats or pseudo-B repeats. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin that comprises only one of one or more A repeats or pseudo-A repeats, or one or more B repeats or pseudo-B repeats. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising one or more A repeats or pseudo-A repeats and one or more B repeats or pseudo-B repeats.

[0016] In some embodiments, the recombinant resilin further comprises a chitin binding domain. In some embodiments, the secreted resilin coding sequence encodes a polypeptide comprising an α mating factor secretion signal. In some embodiments, the secreted resilin coding sequence comprises a FLAG tag.

[0017] In some embodiments, the vector comprises multiple secreted resilin coding sequences. In some embodiments, the vector comprises three secreted resilin coding sequences. In some embodiments, the secreted resilin coding sequence is operably linked to a constitutive or inducible promoter.

[0018] According to some embodiments, recombinant host cells containing one or more vectors comprising a secreted resilin coding sequence are also provided herein. In some embodiments, the recombinant host cell is a yeast cell. In some embodiments, the yeast cell is a methylotrophic yeast cell. In some embodiments, the recombinant host cell is a species selected from the group consisting of Pichia (Komagataella) pastoris, Hansenula polymorpha, Arxula adeninivorans, Yarrowia lipolytica, Pichia (Scheffersomyces) stipitis, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis.

[0019] In some embodiments, the recombinant host cell contains three vectors comprising a secreted resilin coding sequence.

[0020] In some embodiments, the recombinant host cell produces recombinant resilin at a rate greater than 2 mg resilin / g dry cell weight / hour. In some embodiments, the recombinant host cell has a secreted fraction of recombinant resilin, and the secreted fraction is greater than 50%. In some embodiments, the recombinant host cell secretes resilin at a rate greater than 2 mg resilin / g dry cell weight / hour.

[0021] According to some embodiments, a fermentation product is also provided herein, comprising a recombinant host cell containing one or more vectors comprising a secreted resilin coding sequence, and a culture medium suitable for growing the recombinant host cell.

[0022] In some embodiments, the fermentation product contains at least 2 g of recombinant resilin per liter.

[0023] In some embodiments of the fermentation product, more than 80% of the recombinant resilin is present outside the recombinant host cell.

[0024] In some embodiments of the fermentation product, the recombinant resilin is full-length recombinant resilin.

[0025] In some embodiments, compositions are also provided herein, comprising recombinant resilin obtained from a fermentation containing recombinant host cells containing one or more vectors containing secreted resilin coding sequences, and a culture medium suitable for growing the recombinant host cells. In some embodiments, the composition comprises at least 60% by weight of recombinant resilin.

[0026] In some embodiments, the composition has similar properties to a composition containing approximately the same amount of native resilin. In some embodiments, the composition has different properties to a composition containing approximately the same amount of native resilin.

[0027] In some embodiments, the composition contains more than 50% elastic energy. In some embodiments, the composition has a compressive modulus of less than 10 MPa. In some embodiments, the composition has a tensile modulus of less than 10 MPa. In some embodiments, the composition has a shear modulus of less than 1 MPa. In some embodiments, the composition has an extension to break of more than 1%. In some embodiments, the composition has a maximum tensile strength of more than 0.1 kPa. In some embodiments, the composition has a Shore hardness of less than 90. In some embodiments, the composition contains full-length resilience.

[0028] According to some embodiments, methods for preparing compositions comprising recombinant resilin are also provided herein, the method comprising culturing recombinant host cells comprising one or more vectors comprising secretory resilin coding sequences in order to prepare a ferment under conditions that promote the secretion of recombinant resilin from recombinant host cells.

[0029] In some embodiments, a method for preparing a composition containing recombinant resilin further comprises the step of purifying the recombinant resilin to prepare full-length native resilin. In some embodiments, the step of purifying the recombinant resilin to prepare full-length native resilin comprises centrifugation of the ferment to produce a first pellet fraction and a first supernatant fraction, and isolation of recombinant resilin protein from the first pellet fraction. In some embodiments, isolation of recombinant resilin protein from the first pellet fraction comprises adding chaotrope to the first pellet fraction to produce a solution in which the recombinant resilin protein is soluble; centrifugation of the first pellet fraction containing chaotrope to produce a second supernatant fraction and a second pellet fraction, and isolation of recombinant resilin protein from the second supernatant fraction.

[0030] In some embodiments, a method for preparing a composition comprising recombinant resilin further comprises the step of crosslinking multiple recombinant resilin. In some embodiments, the crosslinking is enzymatic crosslinking. In some embodiments, the crosslinking is photochemical crosslinking. In some embodiments, the recombinant resilin protein comprises full-length resilin protein.

[0031] According to some embodiments, a fermentation comprising a culture medium and recombinant host cells is also provided herein, wherein the recombinant host cells comprise a vector, the vector comprises a secretory resilin coding sequence, and the recombinant host cells secrete recombinant resilin at a rate of at least 2 mg / g dry cell weight / hour. [Invention 1001] A step of culturing a population of recombinant host cells in a ferment, wherein the recombinant host cells contain a vector comprising a secretory resilin coding sequence, and the recombinant host cells secrete recombinant resilin proteins encoded by the secretory resilin coding sequence; and A step of purifying the recombinant resilin protein from the fermented product. A method for preparing a composition containing recombinant resilin protein, including the above. [Invention 1002] The method of the present invention 1001, wherein the recombinant resilin protein is a full-length or truncated native resilin. [Invention 1003] The aforementioned native resilin is found in the following insects: Drosophila sechellia, Acromyrmex echinatior, Aeshna, Haematobia irritans, Ctenocephalides felis, Bombus terrestris, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Pediculus humanus corporis, Anopheles gambiae, Glossina morsitans, Atta cephalotes, and Anopheles darlingji. The method of the present invention 1001, derived from an organism selected from the group consisting of darlingi, pea aphid (Acyrthosiphon pisum), black fruit fly (Drosophila virilis), Kirishima fungus fruit fly (Drosophila erecta), sand fly (Lutzomyia longipalpis), giant assassin bug (Rhodnius prolixus), red imported fire ant (Solenopsis invicta), tropical house mosquito (Culex quinquefasciatus), melon fly (Bactrocera cucurbitae), and Trichogramma pretiosum. [Invention 1004] The method of the present invention 1001, wherein the recombinant resilin protein contains SEQ ID NO:1. [Invention 1005] The method of the present invention 1001, wherein the recombinant resilin protein contains SEQ ID NO:4. [Invention 1006] The method of the present invention 1001, wherein the recombinant resilin protein contains an α-conjugation factor secretion signal. [Invention 1007] The method of the present invention 1001, wherein the recombinant resilin protein contains a FLAG tag. [Invention 1008] The method of the present invention 1001, wherein the vector comprises a plurality of secreted resilin coding sequences. [Invention 1009] The method of the present invention 1001, wherein the recombinant host cell is a yeast cell. [Invention 1010] The method of the present invention 1009, wherein the yeast cells are methylotrope yeast cells. [Invention 1011] The method of the present invention 1001, wherein the recombinant host cell is a species selected from the group consisting of Pichia (Komagataella) pastoris, Hansenula polymorpha, Arxula adeninivorans, Yarrowia lipolytica, Pichia (Scheffersomyces) stipitis, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis. [Invention 1012] The method of the present invention 1001, wherein the recombinant host cells produce the recombinant resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour. [Invention 1013] The method of the present invention 1001, wherein the recombinant host cell produces a secreted fraction of the recombinant resilin, and the secreted fraction is greater than 50% of the total recombinant resilin protein expressed by the recombinant host cell. [Invention 1014] The method of the present invention 1001, wherein the recombinant host cells secrete the recombinant resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour. [Invention 1015] The method of the present invention 1001, wherein more than 80% of the recombinant resilin is present outside the recombinant host cells in the fermented product. [Invention 1016] The method of the present invention 1001, wherein the fermented product contains at least 2 g of recombinant resilin per liter. [Invention 1017] The step of purifying the recombinant resilin protein is To produce a first pellet fraction and a first supernatant fraction by centrifuging the fermented product; and Isolating the recombinant resilin protein from the first pellet fraction. The method of the present invention 1001, including the method of the present invention 1001. [Invention 1018] The step of purifying the recombinant resilin protein is To produce a solution in which the recombinant resilin protein is soluble, chaotrope is added to the first pellet fraction; To produce a second supernatant fraction and a second pellet fraction by centrifuging the first pellet fraction containing the chaotrope; and To isolate soluble full-length resilin from the second supernatant fraction. The method of the present invention 1017, further comprising: [Invention 1019] A vector containing a secreted resilin-coding sequence. [Invention 1020] The vector of the present invention 1019, wherein the secreted resilin coding sequence codes for a full-length or truncated native resilin. [Invention 1021] The vector of the present invention 1019, wherein the secreted resilin coding sequence codes for a modified full-length or truncated native resilin. [Invention 1022] The vector of the present invention 1021, wherein the modified resilin comprises the addition of an amino acid residue, the deletion of an amino acid residue, the substitution of an amino acid residue, or the repositioning of an amino acid residue, and the amino acid residue can be crosslinked with another resilin. [Invention 1023] A vector according to any of the invention 1020 to 1022, wherein the full-length or shortened native resilin is derived from an organism selected from the group consisting of Drosophila melanogaster, Leafcutter ants, Aeshna rhinoceros, Stinkfly, Cat flea, Bombus ignitus, Confused flour beetle, Honeybee, Parasitic wasp, Body louse, Anopheles gambia, Glossina moorcitans, Atta cephalotes, Anopheles darlingzi, Pea aphid, Drosophila melanogaster, Drosophila kirishimaensis, Sandfly, Assassin bug, Red imported fire ant, Culex pipiens, Culex tritaeniorhynchus, Trichogramma plethiosum. [Invention 1024] The vector of the present invention 1023, wherein the secreted resilin-coding sequence encodes a polypeptide containing SEQ ID NO:1. [Invention 1025] The vector of the present invention 1023, wherein the secreted resilin coding sequence encodes a polypeptide containing SEQ ID NO:4. [Invention 1026] The vector of the present invention 1019, wherein the secreted resilin coding sequence encodes a recombinant resilin comprising one or more A repeats or quasi-A repeats. [Invention 1027] The vector of the present invention 1019, wherein the secreted resilin coding sequence encodes a recombinant resilin comprising one or more B repeats or quasi-B repeats. [Invention 1028] The vector of the present invention 1019, wherein the secreted resilin coding sequence codes for recombinant resilin, and the recombinant resilin comprises only one or more A repeats or quasi-A repeats, or one or more B repeats or quasi-B repeats. [Invention 1029] The vector of the present invention 1019, wherein the secreted resilin coding sequence codes for recombinant resilin comprising one or more A repeats or quasi-A repeats and one or more B repeats or quasi-B repeats. [Invention 1030] A vector according to any one of the invention 1026 to 1029, further comprising the recombinant resilin and a chitin-binding domain. [Invention 1031] A vector according to any one of the present invention 1019 to 1030, wherein the secreted resilin coding sequence encodes a polypeptide containing an α-conjugation factor secretion signal. [Invention 1032] A vector according to any of the inventions 1019 to 1030, wherein the secreted resilin coding sequence includes a FLAG tag. [Invention 1033] A vector according to any of invention 1019 to 1032, comprising multiple secreted resilin coding sequences. [Invention 1034] A vector according to the present invention 1033, comprising three secreted resilin coding sequences. [Invention 1035] A vector according to any one of the present invention 1019 to 1034, wherein the secreted resilin coding sequence is functionally linked to a constructive or inducible promoter. [Invention 1036] Recombinant host cells comprising one or more vectors according to invention 1019 to 1035. [Invention 1037] A recombinant host cell of the present invention 1036, which is a yeast cell. [Invention 1038] The recombinant host cell of the present invention 1037, wherein the yeast cells are methylotrope yeast cells. [Invention 1039] Recombinant host cells of the present invention 1038, which are species selected from the group consisting of Pichia (Comagatera) pastris, Hanzenula polymorpha, Arcsula adeniniborans, Yarouia liporitica, Pichia (Scheffersomyces) stipityis, Pichia metanorica, Saccharomyces cerevisiae, and Kluiveromyces lactis. [Invention 1040] Recombinant host cells of the present invention 1036, comprising three vectors from any of the present inventions 1019 to 1035. [Invention 1041] Recombinant host cells according to any of the invention 1036-1040, which produce recombinant resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour. [Invention 1042] Recombinant host cells according to any of the invention 1036 to 1041, wherein the recombinant host cell has a secretory fraction of recombinant resilin, and the secretory fraction constitutes more than 50%. [Invention 1043] Recombinant host cells according to any of invention 1036-1042 that secrete resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour. [Invention 1044] A fermented product comprising recombinant host cells according to any of invention 1036 to 1043 and a culture medium suitable for growing the recombinant host cells. [Invention 1045] A fermented product of the present invention 1044, containing at least 2 g of recombinant resilin per liter. [Invention 1046] The fermented product of the present invention 1044, wherein more than 80% recombinant resilin is present outside the recombinant host cell. [Invention 1047] A fermented product according to any one of the present invention 1044 to 1046, wherein the recombinant resilin is full-length recombinant resilin. [Invention 1048] A composition comprising recombinant resilin obtained from any of the fermented products of the present invention 1044 to 1047. [Invention 1049] A composition according to the present invention 1048, comprising at least 60% by weight of recombinant resilin. [Invention 1050] A composition of the present invention 1048 having similar properties to a composition containing approximately the same amount of native resilin. [Invention 1051] A composition of the present invention 1048 having different properties compared to a composition containing approximately the same amount of native resilin. [Invention 1052] A composition of the present invention 1048 having an elastic energy of more than 50%. [Invention 1053] A composition of the present invention 1048 having a compressive modulus of less than 10 MPa. [Invention 1054] A composition of the present invention 1048 having a tensile modulus of less than 10 MPa. [Invention 1055] A composition of the present invention 1048 having a shear modulus of less than 1 MPa. [Invention 1056] A composition according to the present invention 1048 having an extension to break of more than 1%. [Invention 1057] A composition according to the present invention 1048 having a maximum tensile strength of more than 0.1 kPa. [Invention 1058] A composition of the present invention 1048 having a Shore hardness of less than 90. [Invention 1059] A composition of the present invention 1048, comprising full-length resilin. [Invention 1060] A method for producing any of the compositions 1048 to 1059 of the present invention, comprising the step of culturing recombinant host cells of any of the present inventions 1036 to 1042 to produce a ferment under conditions that promote the secretion of recombinant resilin. [Invention 1061] The method of the present invention 1060, further comprising the step of purifying the recombinant resilin in order to produce a full-length native resilin. [Invention 1062] The process of purifying the recombinant resilin in order to produce full-length native resilin is To produce a first pellet fraction and a first supernatant fraction by centrifuging the fermented product; and To isolate recombinant resilin protein from the first pellet fraction. The method of the present invention 1061, including the method of the present invention 1061. [Invention 1063] Isolating the recombinant resilin protein from the first pellet fraction is To produce a solution in which the recombinant resilin protein is soluble, chaotrope is added to the first pellet fraction; To produce a second supernatant fraction and a second pellet fraction by centrifuging the first pellet fraction containing the chaotrope; and Isolating the recombinant resilin protein from the second supernatant fraction. The method of the present invention 1062, including the method of the present invention 1062. [Invention 1064] A method according to any one of the present invention 1060 to 1063, further comprising the step of crosslinking a plurality of the recombinant resilin. [Invention 1065] The method of the present invention 1064, wherein the crosslinking is enzymatic crosslinking. [Invention 1066] The method of the present invention 1064, wherein the crosslinking is photochemical crosslinking. [Invention 1067] The method according to any one of the present invention 1060 to 1066, wherein the recombinant resilin protein comprises a full-length resilin protein. [Invention 1068] A fermented product comprising culture medium and recombinant host cells, A fermentation comprising recombinant host cells comprising a vector, the vector comprising a secretory resilin coding sequence, and the recombinant host cells secreting recombinant resilin at a rate of at least 2 mg / g dry cell weight / hour. [Brief explanation of the drawing]

[0032] [Figure 1] A schematic example of the structure of a representative resilin is shown. [Figure 2] This is a flowchart showing a method for preparing a composition containing recombinant resilin. [Figure 3] This is an exemplary map of a vector containing three secreted resilience-coding sequences. [Figure 4A]This shows the expression and secretion of 3×FLAG-tagged recombinant regirin in recombinant host cells of Pichia pastris (Komagataella phaffii) as assayed by ELISA. [Figure 4B] This image shows recombinant resilin expression in Pichia pastris (Comagatera fafii) recombinant host cells, assayed by Western blotting (top; 3×FLAG-tagged recombinant resilin) ​​and Coomasii (bottom; untagged recombinant resilin). [Figure 5A] This shows the production levels of recombinant resilin from recombinant host cells in a rich medium. [Figure 5B] This shows the production levels of recombinant resilin produced by recombinant host cells in minimal culture medium. [Figure 6] This shows the purification of two secreted recombinant resilin molecules from 500 mL BMGY flask growth. For each sample, lane 1 is the initial supernatant; lane 2 is the supernatant after precipitation; lane 3 is the dialyzed precipitate; lane 4 is the heat-denatured protein; and lane 5 is the final purified recombinant resilin. [Figure 7] Photographs of protein block copolymers containing crosslinked, purified recombinant resilience in various forms and shapes are shown. [Figure 8] A photograph of a compressed protein block copolymer containing crosslinked recombinant resilin is shown. [Figure 9] This is an image of a gel showing bands obtained from a recombinant resilin composition purified by a selected method described herein. [Figure 10] This invention relates to a full-length Seychelles elezirin sequence (Ds_ACB) expressed in conjunction with a signal sequence that is subsequently cleaved, according to several aspects of the present invention. [Modes for carrying out the invention]

[0033] The drawings illustrate various aspects of the present disclosure for illustrative purposes only. Those skilled in the art will readily recognize from the following considerations that alternative embodiments of the structures and methods exemplified herein may be used without departing from the principles described herein.

[0034] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field relating to this disclosure.

[0035] The terms “a,” “an,” and “the,” as well as similar referents, are used herein to mean both singular and plural, unless otherwise indicated herein or explicitly stated in the context.

[0036] The terms “approximately,” “about,” or “similar / nearly the same” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which may depend in part on the method by which the value is measured or determined, or on the limitations of the measuring system. It should be understood that all ranges and quantities below are approximations and are not intended to limit the invention. Where ranges and numbers are used, they may be approximations, including statistical ranges or measurement errors or variability. In some embodiments, for example, the measurement may be ±10%.

[0037] Amino acids can be referred to by one-letter or three-letter codes. The one-letter codes, amino acid names, and three-letter codes are as follows: G - Glycine (Gly), P - Proline (Pro), A - Alanine (Ala), V - Valine (Val), L - Leucine (Leu), I - Isoleucine (Ile), M - Methionine (Met), C - Cysteine ​​(Cys), F - Phenylalanine (Phe), Y - Tyrosine (Tyr), W - Tryptophan (Trp), H - Histidine (His), K - Lysine (Lys), R - Arginine (Arg), Q - Glutamine (Gln), N - Asparagine (Asn), E - Glutamic acid (Glu), D - Aspartic acid (Asp), S - Serine (Ser), T - Threonine (Thr).

[0038] The terms "including," "includes," "having," "has," and "with," or their variations, shall be considered comprehensive, just as the term "comprising."

[0039] The term "microorganism" as used herein refers to microorganisms and single-celled organisms. As used herein, the term includes all bacteria, all archaea, single-celled protists, single-celled animals, single-celled plants, single-celled fungi, single-celled algae, all protozoa, and all Chromista.

[0040] The term “native” means, as used herein, a composition found in nature in its natural, unmodified form.

[0041] The terms “optional” or “optional” mean that a feature or structure may or may not be present, or that an event or situation may or may not occur, and that the description includes cases where a detailed feature or structure is present and cases where a feature or structure is absent, or cases where an event or situation occurs and cases where an event or situation does not occur.

[0042] The term "secretory fraction," as used herein, refers to the fraction of recombinant resilience secreted by cells compared to the total resilience produced by the cells.

[0043] The term "secretion signal," as used herein, refers to a short peptide that, when fused with a polypeptide, mediates the secretion of that polypeptide from the cell.

[0044] The term “secreted resilin coding sequence” means, as used herein, a nucleotide sequence encoding a resilin provided herein, which is fused with a secretory signal at its N-terminus and optionally with a tagged peptide or tagged polypeptide at its C-terminus.

[0045] The term "recombinant" refers, with respect to polypeptides (e.g., resilin), to polypeptides produced in recombinant host cells or polypeptides synthesized from recombinant nucleic acids, as used herein.

[0046] The term "recombinant host cell" as used herein refers to a host cell containing recombinant nucleic acid.

[0047] The term “recombinant nucleic acid” means, as used herein, a nucleic acid isolated from its naturally occurring environment, or a nucleic acid that is not associated with all or part of nucleic acids adjacent to or proximal to it if found in nature, or a nucleic acid functionally linked to a nucleic acid that is not naturally linked, or a nucleic acid that does not exist in nature, or a nucleic acid that contains modifications not found in it in nature (e.g., insertions, deletions, or point mutations introduced artificially, e.g., by human intervention), or a nucleic acid integrated into a chromosome at a heterologous site. The term includes nucleic acids, including cloned DNA isolates and chemically synthesized nucleotide analogs.

[0048] The term “vector,” as used herein, refers to a nucleic acid molecule capable of transporting another ligated nucleic acid. One type of vector is a “plasmid,” which generally refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated, but also includes linear double-stranded molecules such as those obtained by amplification by polymerase chain reaction (PCR) or treatment of a circular plasmid with restriction enzymes. Other vectors include bacteriophages, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another type of vector is a viral vector to which an additional DNA segment can be ligated into a viral genome. Some vectors can autonomously replicate in the cells into which they are introduced (e.g., vectors with replication origins that function in the cell). Other vectors can be incorporated into the cell's genome upon introduction into the cell, thereby replicating together with the cell genome.

[0049] The term "repeat" refers to a subsequence that occurs two or more times within a polynucleotide or polypeptide (e.g., a chain sequence) with respect to an amino acid or nucleic acid sequence, as used herein. A polynucleotide or polypeptide may have direct repeats of a repeat sequence that do not contain an intervening sequence, or it may have discontinuous repeats of a repeat sequence that contain an intervening sequence. The term "quasi-repeat" refers to a subsequence that is inaccurately repeated in a polynucleotide or polypeptide (i.e., some parts of the quasi-repeat subsequence are variable between quasi-repeats) with respect to an amino acid or nucleic acid sequence, as used herein. Repeating polypeptides and DNA molecules (or parts of polypeptides or DNA molecules) may consist of either repeat subsequences (i.e., exact repeats) or quasi-repeat subsequences (i.e., inaccurate repeats).

[0050] The term “native resilin” refers to an elastomer polypeptide or protein produced by an insect, as used herein. GenBank accession numbers for non-limiting examples of native resilins include the following NCBI sequence numbers: NP 995860 (Drosophila melanogaster), NP 611157 (Drosophila melanogaster), Q9V7U0 (Drosophila melanogaster), AAS64829, AAF57953 (Drosophila melanogaster), XP 001817028 (Confused flour beetle), and XP001947408 (Pea aphid).

[0051] The term “modified” means, as used herein, a sequence of a protein or polypeptide that differs in sequence and composition from a native protein or polypeptide, such that its functional properties are conserved within 10% of the properties of the native protein or polypeptide. In some embodiments, the difference between a modified protein or polypeptide and a native protein or polypeptide may be in the primary sequence (e.g., one or more amino acids are removed, inserted, or substituted) or in post-translational modifications (e.g., glycosylation, phosphorylation). Amino acid deletion means the removal of one or more amino acids from a protein. Amino acid insertion means the introduction of one or more amino acid residues into a protein or polypeptide. Amino acid insertions may include fusions of one or more amino acids at the N-terminus and / or C-terminus, as well as intrasequence insertions. Amino acid substitutions include non-conservative and conservative substitutions, and tables of conservative amino acid substitutions are well known in the art (see, for example, Creighton (1984) Proteins. WH Freeman and Company (Eds)). In some embodiments, the amino acid sequence or nucleotide sequence identity between the modified protein or modified polypeptide and the native protein or native polypeptide is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the amino acids or nucleotide bases.

[0052] The term “shortened” means, as used herein, a sequence of a protein or polypeptide that is shorter in length than the native protein or polypeptide. In some embodiments, the shortened protein or polypeptide may be more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the length of the native protein or polypeptide.

[0053] The terms “homologous” or “substantial similarity” refer to polypeptides, nucleic acids, or fragments thereof, and as used herein, indicate that, when optimally aligned with another amino acid or nucleic acid (or its complementary chain) by appropriate amino acid or nucleotide insertion or deletion, there is amino acid or nucleotide sequence identity in at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the amino acid or nucleotide bases, as measured by well-known algorithms of sequence identity such as FASTA, BLAST, or Gap as described above.

[0054] The term “resilin” as used herein refers to a protein or polypeptide that can be crosslinked to form an elastomer, where the protein or polypeptide is a native resilin, a modified native resilin, or a truncated native resilin. The resilin of the present invention is preferably a recombinant resilin. In some embodiments, the recombinant resilin contains a native or modified (e.g., truncated or chained) nucleotide sequence encoding a resilin or resilin fragment (e.g., isolated from an insect) heterologously expressed and secreted from a host cell. In a preferred embodiment, the secreted recombinant resilin protein is collected from an extracellular solution of a host cell.

[0055] As used herein, the term “elastomer” refers to a polymer that is viscoelastic and (except for covalent intermolecular crosslinks, if present) typically has weak intermolecular forces. Viscoelasticity is a property of materials that exhibits both viscous and elastic characteristics when deformed, and therefore exhibits time-dependent strain. Elasticity is related to the expansion and contraction of bonds along crystal planes in an ordered solid, while viscosity is a result of the diffusion of atoms or molecules within an amorphous material. Thus, elastomers that are viscoelastic generally have a lower Young's modulus and a higher fracture strain compared to other materials. Due to the viscous component of the material, when a load is applied and then removed, the energy of the viscoelastic material dissipates. This phenomenon is observed as hysteresis in the stress-strain curve of a viscoelastic material. When a load is applied, a specific stress-strain curve exists, and when the load is removed, the stress-strain curve at unloading is different from the curve at loading. The dissipated energy is the area between the loading curve and the unloading curve.

[0056] The enumeration of value ranges in this specification is merely a shorthand notation for referring individually to each separate value that is comprehensively included within that range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually enumerated.

[0057] Detailed explanation Compositions containing recombinant resilin and methods for preparing them are provided herein.

[0058] Resilin possesses many unique properties compared to petroleum-based elastomers. Most notably, resilin has extremely high elastic efficiency, meaning that the energy input for deformation is hardly lost as heat. Other desirable properties of resilin include, for example, desirable elastic energy, compressive modulus, tensile modulus, shear modulus, elongation at break, maximum tensile strength, hardness, rebound, and compression set. Furthermore, resilin is a protein and therefore biodegradable, making it more environmentally friendly than petroleum-based polymers. Resilin is also biocompatible and can therefore be used in applications involving contact with humans or animals. Finally, it is possible to tune the mechanical properties of recombinant resilin by varying the protein sequence, protein structure, amount of intermolecular crosslinking, and working variables to create elastomers designed for specific application areas.

[0059] In some embodiments, the methods and compositions provided herein provide efficient means for producing large quantities of recombinant resilin. In some embodiments, large quantities of resilin and resilin-like polypeptides are obtained using recombinant host cells that secrete recombinant resilin via a secretory pathway. Such secretion of recombinant resilin (a) avoids toxicity from intracellular accumulation of recombinant resilin, (b) simplifies purification by eliminating the process of cell disruption or protein refolding, and (c) provides opportunities for posttranslational events (e.g., maturation by proteolysis, glycosylation, disulfide bond formation) that can modulate the properties of recombinant resilin.

[0060] Composition containing recombinant resilin In some embodiments, the compositions provided herein include recombinant resilin.

[0061] Figure 1 illustrates an example of a native resilin containing an N-terminal A domain with multiple repeat units ("A repeats") containing the consensus amino acid sequence YGXP where X is any amino acid; a chitin-bound RR-2(C) domain (Pfam reference PF00379; Rebers JE & Willis, JH. A conserved domain in anthropod cuticular proteins binds chitin. Insect Biochem Mol Biol 31:1083-1093); and a C-terminal B domain with multiple repeat units ("B repeats") containing the consensus amino acid sequence UYZXZ where U is glycine or serine; Z is serine, glycine, arginine, or proline; and X is any amino acid. Not all naturally occurring resilins possess A, C, and B domains. Native resilins produced by various insects typically have inaccurate repeats (i.e., quasi-repeats) with some amino acid variations between quasi-repeats within the A domain and / or B domain.

[0062] In some embodiments, recombinant resilin provided herein comprises one or more A repeats. In some embodiments, recombinant resilin comprises an N-terminal A domain comprising a plurality of blocks of amino acid subsequences of A repeats and / or quasi-A repeats, each having the consensus sequence SXXYGXP, where S is serine, X is an amino acid, Y is tyrosine, G is glycine, and P is proline.

[0063] In some embodiments, recombinant resilin provided herein comprises one or more B repeats. In some embodiments, recombinant resilin comprises a C-terminal B domain comprising a plurality of blocks of amino acid subsequences of B repeats and / or quasi-B repeats, each having the consensus sequence GYZXZZX and / or SYZXZZX, where G is glycine; Y is tyrosine; Z is serine, glycine, proline, or arginine; S is serine; and X is any amino acid.

[0064] In some embodiments, recombinant resilin provided herein comprises one or more A repeats. In some such embodiments, recombinant resilin comprises 1 to 100 A repeats, or 2 to 50 A repeats, or 5 to 50 A repeats, or 5 to 20 A repeats.

[0065] In some embodiments, recombinant resilin is given by formula: (X1-X2-X3-X4) n (1) Includes one or more consensus sequences described by In the formula, parentheses indicate the boundaries of repeats or quasi-repeats in the consensus sequence; n indicates the number of A repeats or quasi-A repeats, which can be 1-100, 2-50, 5-50, or 5-20; X1 is a motif with a length of 4 amino acids, where the first amino acid of X1 is Y, and the remaining amino acids of X1 are GAP, GLP, GPP, GTP, or GVP; X2 is a motif with a length of 3 to 20 amino acids; X2 contains GGG, GGGG, N, NG, NN, NGN, NGNG, GQGG, GQGN, GQGQ, GQGQG, or three or more glycine residues, or 50% or more of the residues of X2 are either glycine or asparagine, or 60% or more of the residues of X2 are either glycine or asparagine, or 70% or more of the residues of X2 are either glycine or asparagine, or 80% or more of the residues of X2 are either glycine or asparagine; X3 is a motif with a length of 2-6 amino acids, and X3 is GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG; and X4 is a motif with a length of 1-2 amino acids, and X4 can be S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS.

[0066] In some such embodiments, recombinant resilin comprises motifs X1, X2, X3, and X4, and in other embodiments, recombinant resilin comprises motifs X1, X2, X3, or X4, or a combination thereof.

[0067] In some embodiments, recombinant resilin provided herein comprises one or more B repeats. In some such embodiments, recombinant resilin comprises 1 to 100 B repeats or 2 to 50 A repeats or 5 to 50 A repeats or 5 to 20 A repeats.

[0068] In some embodiments, recombinant resilin is given by formula: (X 11 -X 12 -X 13 ) m (2) Includes one or more consensus sequences described by In the formula, parentheses indicate the boundaries of repeats or quasi-repeats in the consensus sequence; m represents the number of B repeats or quasi-B repeats, ranging from 1 to 100; X 11 It is a motif with a length of 1 to 5 amino acids, where the first amino acid is Y, and the remaining amino acids often include GAP, GPP, SSG, or SGG; X 12 It is a motif with a length of 2-5 amino acids and includes GQ, GN, RPG, RPGGQ, RPGGN, SSS, SKG, or SN; and X 13 This is a motif with a length of 4 to 30 amino acids, and contains GG, DLG, GFG, GGG, RDG, SGG, SSS, GGSF, GNGG, GGAGG, or three or more glycine residues, or 30% or more of the residues are glycine, or 40% or more of the residues are glycine, or 50% or more of the residues are glycine, or 60% or more of the residues are glycine.

[0069] In some such embodiments, recombinant resilin comprises motif X 11 , X 12 , and X 13 ; and in other such embodiments, recombinant resilin comprises motif X 11 , X 12 , or X 13 , or combinations thereof.

[0070] In some embodiments, the recombinant resilin provided herein comprises one or more A repeats, one or more B repeats, and / or one or more C domains. In some embodiments, recombinant resilin comprises only one of one or more A repeats or one or more B repeats. In some embodiments, recombinant resilin comprises one or more A repeats and neither B repeats nor C domains. In some embodiments, recombinant resilin comprises one or more B repeats and neither A repeats nor C domains. In embodiments where recombinant resilin comprises a C domain, the C domain may be located on the N-terminal side or C-terminal side of an A repeat or B repeat, or between an A repeat and a B repeat.

[0071] In some embodiments, recombinant resilin further comprises the sequence XXEPPVSYLPPS where X is any amino acid. In some such embodiments, the sequence is located on the N-terminal side of an A repeat or B repeat.

[0072] In some embodiments, recombinant resilin is a full-length native resilin expressed in a non-native environment. In some embodiments, recombinant resilin includes a shortened version of the native resilin. In some embodiments, the shortened native resilin includes at least one A repeat. In some embodiments, the shortened native resilin includes at least one B repeat. Non-limiting examples of full-length and shortened native resilins are provided as SEQ ID NO:1-44. In some embodiments, recombinant resilin is full-length Seychelles drosophila resilin (SEQ ID NO:1). In some embodiments, recombinant resilin is shortened Panama hakiria resilin (SEQ ID NO:4). In some embodiments, recombinant resilin is a full-length or shortened native resilin crosslinked in a non-native manner (e.g., fewer or more crosslinks, crosslinks via different amino acid residues).

[0073] In some embodiments, recombinant resilin is a modified full-length or truncated native resilin. In some embodiments, recombinant resilin is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to full-length native resilin or truncated native resilin. In some embodiments, recombinant resilin is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to full-length Seychelles elegiline (SEQ ID NO:1). In some embodiments, recombinant resilin is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to truncated Panama leafcutter resilin (SEQ ID NO:4).

[0074] Numerous different algorithms that can be used to measure the identity of nucleotide or protein sequences are known in the art. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs in the Wisconsin Package Version 10.0 (Genetics Computer Group (GCG), Madison, Wis). FASTA provides alignment of the region of best overlap between the query sequence and the search sequence, and percentage sequence identity. See, for example, Pearson, Methods Enzymol. 183:63-98, 1990 (the whole of which is incorporated herein by reference). For example, percentage sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (word size of 6 and NOPAM factor for the scoring matrix), or using Gap with the default parameters provided in GCG version 6.1, which is incorporated herein by reference. Alternatively, the sequences may be compared using the computer program BLAST (Altschul et al., J.Mol.Biol.215:403-410, 1990; Gish and States, Nature Genet.3:266-272, 1993; Madden et al., Meth.Enzymol.266:131-141, 1996; Altschul et al., Nucleic Acids Res.25:3389-3402, 1997; Zhang and Madden, Genome Res.7:649-656, 1997), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res.25:3389-3402, 1997).

[0075] In some embodiments, the modified resilin differs from full-length native resilin or shortened native resilin in terms of post-translational modifications (e.g., phosphorylation, glycosylation) at amino acid residues, such that it has one or more post-translational modifications at different positions and / or in different amounts and / or of different types than full-length native resilin or shortened native resilin. In some embodiments, the modified resilin differs from full-length native resilin or shortened native resilin in terms of crosslinking amino acid residues, such that it has one or more crosslinking amino acids at different positions and / or in different amounts and / or of different types than full-length native resilin or shortened native resilin. In some such embodiments, the modified resilin differs from full-length native resilin or shortened native resilin in that it contains one or more additional or fewer tyrosine residues, one or more additional or fewer lysine residues, and / or one or more additional or fewer cysteine ​​residues.

[0076] In some embodiments, recombinant resilin comprises a chain of native resilin or a truncated native resilin or a chain of modified resilin. In some embodiments, the chain of native resilin or the truncated native resilin or the chain of modified resilin comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) A repeats. In some embodiments, the chain of truncated native resilin or the chain of modified resilin comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) B repeats.

[0077] The compositions provided herein contain, by weight, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%; 10% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or This includes 20%; 20%~100%, 90%, 80%, 70%, 60%, 50%, 40%, or 30%; 30%~100%, 90%, 80%, 70%, 60%, 50%, or 40%; 40%~100%, 90%, 80%, 70%, 60%, or 50%; 50%~100%, 90%, 80%, 70%, or 60%; 60%~100%, 90%, 80%, or 70%; 70%~100%, 90%, or 80%; 80%~100% or 90%; or 90%~100% recombinant resilin. The recombinant resilin may be the same recombinant resilin or a mixture of recombinant resilin having at least two different amino acid sequences.

[0078] In some embodiments, the compositions provided herein have properties similar to those of compositions containing native resilin. In other embodiments, the compositions provided herein have properties different from those of compositions containing native resilin. Non-limiting examples of such properties include elastic energy, compressive modulus, tensile modulus, shear modulus, elongation at break, maximum tensile strength, hardness, rebound, and compression set. Parameters that can be modified to obtain compositions having specific mechanical properties include, for example, the length and / or arrangement of recombinant resilin, the degree and / or type of post-translational modification of recombinant resilin, and / or the degree and / or type of crosslinking of recombinant resilin.

[0079] In some embodiments, mechanical properties such as maximum tensile strength, compressive modulus, tensile modulus, shear modulus, elongation at break, and elastic energy can be measured using various types of tensile and compressive systems that perform stress-strain measurements on elastomer samples. The resulting stress-strain curves, including curves with hysteresis, can be measured in tension or compression. In some embodiments, tensile and compressive testing systems can apply strain to a sample and measure the resulting force using a load cell. In some embodiments, mechanical properties may be measured on a macroscopic scale (e.g., using a macroscopic compression tester), a microscopic scale, or a nanoscale (e.g., using atomic force microscopy (AFM) or nanoindentation measurement). In some embodiments, the compressive mechanical properties of elastomers can be measured by the standard ASTM D575-91 (2012) Standard Test Methods for Rubber Properties in Compression. Mechanical measurements of elastomers in tension may be performed using ASTM D412-15a Standard Test Methods for Vulcanized Rubber and Thermoplastic Elastomers—Tension. In some embodiments, the tear strength of elastomers may be performed using ASTM D624-00 Standard Test Method for Tear Strength of Conventional Vulcanized Rubber and Thermoplastic Elastomers.In some embodiments, the mechanical properties of slab elastomers, bonded elastomers, and molded elastomers may be performed using ASTM D3574-11 Standard Test Methods for Flexible Cellular Materials—Slab, Bonded, and Molded Urethane Foams. In some embodiments, the mechanical properties of elastomers may be measured using ASTM D5992-96 (2011) Standard Guide for Dynamic Testing of Vulcanized Rubber and Rubber-Like Materials Using Vibratory Methods.

[0080] In some embodiments, the compositions provided herein have an elastic energy of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%; 50% to 100%, 90%, 80%, 70%, or 60%; 60% to 100%, 90%, 80%, or 70%; 70% to 100%, 90%, or 80%; 80% to 100%, or 90%; 90% to 100%; 95% to 100%, 90% to 99%, or 95% to 99%.

[0081] In some embodiments, the compositions provided herein have a compressive modulus of less than 10 MPa, less than 7 MPa, less than 5 MPa, less than 2 MPa, less than 1 MPa, less than 0.5 MPa, or less than 0.1 MPa; 0.01 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, 1 MPa, 0.5 MPa, or 0.1 MPa; 0.1 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, 1 MPa, or 0.5 MPa; 0.5 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, or 1 MPa; 1 MPa to 10 MPa, 7 MPa, 5 MPa, or 2 MPa; 2 MPa to 10 MPa, 7 MPa, or 5 MPa; 5 MPa to 10 MPa or 7 MPa; or 7 MPa to 10 MPa. In some embodiments, the compressive modulus of the composition may be measured as defined by ASTM D575-91 (2012) Standard Test Method for Rubber Properties in Compression.

[0082] In some embodiments, the compositions provided herein have a tensile modulus of less than 10 MPa, less than 7 MPa, less than 5 MPa, less than 2 MPa, less than 1 MPa, less than 0.5 MPa, or less than 0.1 MPa; 0.01 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, or 0.5 MPa; 0.5 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, or 1 MPa; 1 MPa to 10 MPa, 7 MPa, 5 MPa, or 2 MPa; 2 MPa to 10 MPa, 7 MPa, or 5 MPa; 5 MPa to 10 MPa or 7 MPa; or 7 MPa to 10 MPa.

[0083] In some embodiments, compositions provided herein have a shear modulus of less than 1 MPa, less than 100 kPa, less than 50 kPa, less than 20 kPa, less than 10 kPa, or less than 1 kPa; 0.1 kPa to 1 MPa, 100 kPa, 50 kPa, 20 kPa, 10 kPa, or 1 kPa; 1 kPa to 1 MPa, 100 kPa, 50 kPa, 20 kPa, or 10 kPa; 10 kPa to 1 MPa, 100 kPa, 50 kPa, or 20 kPa; 20 kPa to 1 MPa, 100 kPa, or 50 kPa; 50 kPa to 1 MPa or 100 kPa; or 100 kPa to 1 MPa.

[0084] In some embodiments, the compositions provided herein have elongations at break of more than 1%, more than 10%, more than 50%, more than 100%, more than 300%, or more than 500%; 1% to 500%, 300%, 100%, 50%, or 10%; 10% to 500%, 300%, 100%, or 50%; 50% to 500%, 300%, or 100%; 100% to 500%, or 300%; or 300% to 500%.

[0085] In some embodiments, compositions provided herein have a maximum tensile strength of greater than 0.1 kPa, greater than 1 kPa, greater than 2 kPa, greater than 5 kPa, or greater than 10 kPa; 0.1 kPa to 100 kPa, 10 kPa, 5 kPa, 2 kPa, or 1 kPa; 1 kPa to 100 kPa, 10 kPa, 5 kPa, or 2 kPa; 2 kPa to 100 kPa, 10 kPa, or 5 kPa; 5 kPa to 100 kPa or 10 kPa; or 10 kPa to 100 kPa.

[0086] In some embodiments, mechanical properties such as hardness and compressive modulus can be measured using indentation measurement systems and nanoindentation measurement systems. In some embodiments, indentation measurement, which uses an indenter to indent a sample to a predetermined amount of strain, is used to measure the hardness and compressive modulus of resilin, and the resulting force is measured using a load cell. In some embodiments, various indenter types, including Vickers-type and Berkovich-type indenters, can be used. In some embodiments, the hardness measured by the indentation technique is characterized by the relationship hardness = (peak force) / (contact area).

[0087] In some embodiments, the hardness of polymers, elastomers, and rubbers can be measured using a durometer. In some embodiments, the hardness of elastomers can be measured using the standard ASTM D2240, which recognizes 12 different durometer scales using a specific combination of spring force and indenter shape. The most common scales are the Shore 00, A, and D hardness scales. The hardness scale ranges from 0 to 100, with 0 being a more flexible material and 100 being a harder material.

[0088] In some embodiments, the compositions provided herein have a Shore hardness of less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, or less than 20; 10 to 90, 80, 70, 60, 50, 40, 30, or 20; 20 to 90, 80, 70, 60, 50, 40, or 30; 30 to 90, 80, 70, 60, 50, or 40; 40 to 90, 80, 70, 60, or 50; 50 to 90, 80, 70, or 60; 60 to 90, 80, or 70; 70 to 90 or 80; or 80 to 90. In some embodiments, the hardness measurement of resilin is carried out according to ASTM D2240.

[0089] As used herein, the term “rebound” refers to a detailed measure of elastic energy. In some embodiments, rebound can be measured by a number of different tools, including pendulum tools and dropped balls. In pendulum-type measurements, the rate of rebound, commonly called RB, is given by the formula: It can be obtained from TIFF0007856997000001.tif10128. The rebound elastic energy is, It can be calculated as TIFF0007856997000002.tif9128, where h = peak height of rebound and H = initial height. The rebound elastic energy can also be determined by measuring the rebound angle. Some examples of test methods for determining the rebound of elastomers are ASTM D2632-15 and ASTM D7121-05 (2012).

[0090] In some embodiments, the compositions provided herein have rebounds of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%; 50% to 100%, 90%, 80%, 70%, or 60%; 60% to 100%, 90%, 80%, or 70%; 70% to 100%, 90%, or 80%; 80% to 100% or 90%; 90% to 100%; 95% to 100%, 90% to 99%, or 95% to 99%. In some embodiments, the rebound measurement of resilin is carried out according to ASTM D2632-15 or ASTM D7121-05 (2012).

[0091] As used herein, the term “compression set” refers to a measure of permanent deformation remaining after stress has been removed. In some embodiments, compression set can be measured in different ways, including compression set due to a constant force in air (referred to as compression set A), compression set due to a constant deflection in air (referred to as compression set B), and compression set due to a constant deflection in air taking into account the stiffness of the material (referred to as compression set C). A ) is given by the following formula: C A =[(t0-ti It is calculated by [(thickness of the first sample) / t0] × 100, where t0 is the thickness of the first sample and t i This is the thickness of the sample after testing. Compression set B(C) B ) is C B =[(t0-t i ) / (t0-t n ) × 100 is given by, where t0 is the thickness of the first sample, and t i is the thickness of the sample after testing, and t n is the thickness of the spacer or specimen during the test. Some examples of test methods for determining the compression set of elastomers are ASTM D3574-11 and ASTM D395-16.

[0092] In some embodiments, the compositions provided herein have a compression set A or compression set B of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%; 50% to 100%, 90%, 80%, 70%, or 60%; 60% to 100%, 90%, 80%, or 70%; 70% to 100%, 90%, or 80%; 80% to 100% or 90%; 90% to 100%; 95% to 100%, 90% to 99%, or 95% to 99%. In some embodiments, the compression set measurement of resilin is carried out according to ASTM D3574-11 and ASTM D395-16.

[0093] The processing and molding of resilin into products can take many forms for various applications. Accordingly, the compositions provided herein may have any form and shape, including but not limited to gels, porous sponges, films, machinable solids, cast forms, molded forms, and compounds.

[0094] The compositions provided herein have numerous uses, including, but not limited to, applications in aerospace, automotive, sporting goods, vibration isolation, footwear, and clothing. Several applications from these categories are listed as non-limiting examples. For desired elastic efficiency, Resilin can be used as an energy storage device (e.g., rubber band) for the storage and recovery of mechanical energy. Automotive suspension systems can be improved by applying Resilin bushings to maintain more contact between the tire and the road when crossing bumps and depressions at high speeds. Furthermore, there are numerous sporting goods applications of Resilin with differently tuned mechanical properties, including golf ball cores, tennis racket grips, golf club grips, and table tennis rackets.

[0095] Due to the unique properties of the Resilin compositions provided herein, the applications of particular interest are footwear. As insoles or midsoles, Resilin can improve the comfort and biological efficiency of shoes by mitigating ground contact and recovering more energy from ground contact as forward propulsion. As midsoles, Resilin may constitute the entire midsole or be embedded within another material (e.g., a material resistant to abrasion or wear, or a material tuned for traction) to complement its properties. A Resilin midsole may contain multiple Resilin materials having differently tuned mechanical properties that work together to provide enhanced performance (e.g., a more flexible heel strike area and firmer arch support).

[0096] As used herein, the term “density” refers to the mass of a sample divided by its volume. In some embodiments, the density of an elastomer may be determined using a specific gravity bottle containing alcohol instead of water to eliminate air bubbles. In some embodiments, the density of an elastomer may be determined using a hydrostatic method. As used herein, “compressed volume density” refers to the ratio of the mass of a sample to its compressed volume, where “compressed volume” is defined as the final equilibrium volume reached by the elastomer sample when subjected to sufficient compressive force to flow until it perfectly conforms to the shape around a piston cylinder test chamber enclosure. In some embodiments, the compressed volume density of an elastomer may be determined using a compressed volume density meter.

[0097] In some embodiments, the compositions provided herein contain 0.5 mg / cm³ 3 ~2.0 mg / cm³ 3 or 1.0 mg / cm³ 3 ~1.5 mg / cm³ 3 or 1.1 mg / cm³ 3 ~1.4 mg / cm³ 3 or 1.2 mg / cm³ 3 ~1.35 mg / cm³ 3 It has a density or compressed volume density. In some embodiments, the determination of the density or compressed volume density of the elastomer can be carried out using ASTM D297-15 Standard Test Methods for Rubber Products-Chemical Analysis.

[0098] Recombinant resilience vectors, recombinant host cells, and fermented products Vectors encoding recombinant resilin, recombinant host cells containing such vectors, and fermentations containing such recombinant host cells and recombinant resilin are further provided herein.

[0099] In some embodiments, the vectors provided herein include a secreted resilin coding sequence encoding a resilin polypeptide fused to a secretory signal at its N-terminus and optionally fused to a tag peptide or tag polypeptide at its C-terminus. In some embodiments, the vector includes a secreted resilin coding sequence that is codon-optimized for expression in specific host cells.

[0100] Appropriate secretory signals are those that mediate the secretion of polypeptides in recombinant host cells provided herein. Non-limiting examples of appropriate secretory signals include the α-junction factor (αMF) of Saccharomyces cerevisiae, the acid phosphatase (PHO1) of Pichia pastoris, and the secretory signal of phytohemagglutinin (PHA-E) derived from the common bean, kidney bean (Phaseolus vulgaris). Additional secretory signals may be known in the art or may be identified by the identification of proteins secreted by host cells, followed by genomic analysis of the secreted proteins and identification of the untranslated N-terminal sequence (see, for example, Huang et al. A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures. Appl Microbiol Biotechnol. 2011 Apr;90(1):235-47).

[0101] The resilin encoded by the secreted resilin coding sequence may be further fused with a tag peptide or tag polypeptide. Non-limiting examples of tag peptides or tag polypeptides include affinity tags (i.e., peptides or polypeptides that bind to certain agents or matrices), solubilization tags (i.e., peptides or polypeptides that assist in the proper folding of proteins and prevent precipitation), chromatography tags (i.e., peptides or polypeptides that alter the chromatographic properties of a protein to give different separations between specific separation techniques), epitope tags (i.e., peptides or polypeptides to which antibodies bind), fluorescent tags (i.e., peptides or polypeptides that emit high-wavelength light when excited by short-wavelength light), chromogenic tags (i.e., peptides or polypeptides that absorb specific segments of the visible light spectrum), enzyme substrate tags (i.e., peptides or polypeptides that are substrates for specific enzymatic reactions), chemical substrate tags (i.e., peptides or polypeptides that are substrates for specific chemical modifications), or combinations thereof. Non-limiting examples of appropriate affinity tags include maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) tags, SBP tags, Strep tags, and calmodulin tags. Non-limiting examples of appropriate soluble tags include thioredoxin (TRX), poly(NANP), MBP, and GST. Non-limiting examples of chromatographic tags include polyanionic amino acids (e.g., This includes TIFF0007856997000003.tif4128) and polyglutamate tags. Non-limiting examples of epitope tags include V5 tags, VSV tags, Myc tags, HA tags, E tags, NE tags, and FLAG tags. Non-limiting examples of fluorescent tags include green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), orange fluorescent protein (OFP), red fluorescent protein (RFP), and their derivatives. Non-limiting examples of chromogenic tags include non-fluorescent members of the GFP-like protein family (e.g., BlitzenBlue, DonnerMagenta; DNA2.0, Neward, CA). Non-limiting examples of enzyme substrate tags include peptides or polypeptides containing lysine suitable for biotinylation in their sequence (e.g., AviTag, biotin carboxyl carrier protein [BCCP]). Non-limiting examples of chemical substrate tags include substrates suitable for reaction with FIAsH-EDT2. The fusion of the C-terminal peptide or polypeptide with the resilin may or may not be cleavable (e.g., by TEV protease, thrombin, factor Xa, or enteropeptidase).

[0102] In some embodiments, the vector comprises one secretory resilin-coding sequence. In other embodiments, the vector comprises two or more (e.g., three, four, or five) secretory resilin-coding sequences. In some such embodiments, the secretory resilin-coding sequences are identical. In other such embodiments, at least two of the secretory resilin-coding sequences are not identical. In embodiments where at least two of the secretory resilin-coding sequences are not identical, at least two of the secretory resilin-coding sequences may be different from each other in the resilins and / or secretory signals and / or any tag peptides or tag polypeptides they encode.

[0103] In some embodiments, the vector includes a promoter functionally linked to a secreted resilin coding sequence to drive the expression of the secreted resilin coding sequence. The promoter may be a constitutive promoter or an inducible promoter. In some embodiments, induction of an inducible promoter occurs via glucose repression, galactose repression, sucrose repression, phosphate repression, thiamine repression, or methanol repression. Suitable promoters include those that mediate protein expression in recombinant host cells provided herein. Non-limiting examples of suitable promoters include the AOX1 promoter, GAP promoter, LAC4-PBI promoter, T7 promoter, TAC promoter, GCW14 promoter, GAL1 promoter, λPL promoter, λPR promoter, β-lactamase promoter, spa promoter, CYC1 promoter, TDH3 promoter, GPD promoter, TEF1 promoter, ENO2 promoter, PGL1 promoter, SUC2 promoter, ADH1 promoter, ADH2 promoter, HXT7 promoter, PHO5 promoter, and CLB1 promoter. Additional promoters that can be used to facilitate the expression of secreted resilin-coding sequences are known in the art.

[0104] In some embodiments, the vector includes a terminator functionally ligated to a secretory resilin coding sequence to result in the termination of transcription of the secretory resilin coding sequence. Suitable terminators include those that terminate transcription in recombinant host cells provided herein. Non-limiting examples of suitable terminators include the AOX1 terminator, the PGK1 terminator, and the TPS1 terminator. Additional terminators that result in the termination of transcription of secretory resilin coding sequences are known in the art.

[0105] In embodiments in which the vector comprises two or more resilin coding sequences, the two or more resilin coding sequences may be functionally ligated to the same promoter and / or terminator, or to two or more different promoters and / or terminators.

[0106] The vectors provided herein may further include elements suitable for vector propagation in recombinant host cells. Non-limiting examples of such elements include bacterial replication origins and selection markers (e.g., antibiotic resistance genes, nutrient requirement markers). Bacterial replication origins and selection markers are known in the art. In some embodiments, the selection marker is a drug resistance marker. A drug resistance marker allows cells to detoxify exogenously added drugs that would otherwise kill the cells. Exemplary examples of drug resistance markers include, but are not limited to, those relating to resistance to antibiotics such as ampicillin, tetracycline, kanamycin, bleomycin, streptomycin, hygromycin, neomycin, and Zeocin®. In some embodiments, the selection marker is a nutrient requirement marker. A nutrient requirement marker allows cells to synthesize their essential components (generally amino acids) while being grown in a medium lacking those essential components. Selectable nutrient requirement gene sequences include, for example, hisD, which enables growth in histidine-free medium in the presence of histidinol. Other select markers suitable for the vector of the present invention include bleomycin resistance genes, metallothionein genes, hygromycin B phosphotransferase genes, AURI genes, adenosine deaminase genes, aminoglycoside phosphotransferase genes, dihydrofolate reductase genes, thymidine kinase genes, and xanthine guanine phosphoribosyltransferase genes.

[0107] The vector of the present invention may further include a targeting sequence that directs the incorporation of a secreted resilin-coding sequence to a specific location within the genome of a host cell. Non-limiting examples of such a targeting sequence include a nucleotide sequence identical to a nucleotide sequence present in the genome of the host cell. In some embodiments, the targeting sequence is identical to a repeating element within the genome of the host cell. In some embodiments, the targeting sequence is identical to a transposition element within the genome of the host cell.

[0108] In some embodiments, recombinant host cells containing the vectors described herein are provided herein. In some embodiments, the vectors are stably integrated into the genome (e.g., chromosomes) of the recombinant host cell, for example, via homologous recombination or targeted integration. Non-limiting examples of suitable sites for genomic integration include the Ty1 locus in the Saccharomyces cerevisiae genome, the rDNA locus and HSP82 locus in the Pichia pastris genome, and transposition elements having copies scattered throughout the genome of the recombinant host cell. In other embodiments, the vectors are not stably integrated into the genome of the recombinant host cell and exist outside the chromosomes.

[0109] Recombinant host cells may originate from mammals, plants, algae, fungi, or microorganisms. Non-limiting examples of suitable fungi include methylotrope yeast, filamentous yeast, Arcsula adeniniborans, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida liporitica, Candida pseudotropicalis, Candida utilis, Candida versatilis, Debaryomyces hansenii, and Endothia (a type of chestnut blight fungus). parasitica), Eremothecium ashbyii, Fusarium moniliforme, Hanzenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor miehei var. Cooney et EmersonCooney et Emerson), Mucor pusillus Lindt, Penicillium roquefortii, Pichia metanorica, Pichia pastris (Comagatera faffii), Pichia (Scheffersomyces) stipitis, Rhizopus niveus, Rhodotorula, Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces diastaticus, Saccharomyces ellipsoides (Saccharomyces Saccharomyces ellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum, Sporidiobolus johnsonii, Sporidiobolus salmonicolor, Sporobolomyces roseus, Trichoderma liesei This includes *Xanthophyllomyces dendrorhous*, *Yarrowia lipopolitica*, *Zygosaccharomyces rouxii*, and their derivatives and hybrids.

[0110] Non-limiting examples of suitable microorganisms include Acetobacter suboxydans, Acetobacter xylinum, Actinoplane missouriensis, Arthrospira platensis, Arthrospira maxima, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, Escherichia coli, and Lactobacillus acidophilus. acidophilus), Lactobacillus bulgaricus, Lactobacillus reuteri, Lactococcus lactis, Lactococcus lactis Lancefield N group, Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroides NRRL B-512(F) strain, Micrococcus lysodeikticus, Spirulina, Streptococcus cremoris Streptococcus cremoris), Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilusThis includes Streptomyces thermophilus, Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes, Streptomyces rubiginosus, Xanthomonas campestris, and their derivatives and hybrids. Additional strains that can be used as recombinant host cells are known in the art. It should be understood that the term “recombinant host cell” refers not only to specific principal cells but also to the offspring of such cells. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such offspring may not actually be identical to the parent cells, but they are still included within the scope of the term “recombinant host cell” as used herein.

[0111] In some embodiments, recombinant host cells include genetic modifications that enhance the production of recombinant resilin provided herein. Non-limiting examples of such genetic modifications include altered promoters, altered kinase activity, altered protein folding activity, altered protein secretion activity, altered gene expression induction pathways, and altered protease activity.

[0112] Recombinant host cells provided herein are produced by transforming cells of appropriate origin using vectors provided herein. The vectors for such transformation may be circular or linear. Recombinant host cell transformants containing the vector can be readily identified, for example, by expressing a drug resistance marker or nutritional requirement marker encoded by the vector, which allows for selection based on the ability of the cells to proliferate, or by other means (e.g., detection of luminescent peptides contained in the vector, e.g., restriction enzyme mapping, PCR amplification, or molecular analysis of individual recombinant host cell colonies by sequence analysis of isolated extrachromosomal vectors or chromosomal integration sites).

[0113] In some embodiments, recombinant host cells provided herein can produce high titers of recombinant resilin provided herein. In some such embodiments, recombinant host cells are at concentrations of 2 mg resilin / g dry cell weight / hour, 4 mg resilin / g dry cell weight / hour, 6 mg resilin / g dry cell weight / hour, 8 mg resilin / g dry cell weight / hour, 10 mg resilin / g dry cell weight / hour, 12 mg resilin / g dry cell weight / hour, 14 mg resilin / g dry cell weight / hour, 16 mg resilin / g dry cell weight / hour, 18 mg resilin / g dry cell weight / hour, 20 mg resilin / g dry cell weight / hour, 25 mg resilin / g dry cell weight / hour, or above 30 mg resilin / g dry cell weight / hour; 2-40, 30, 20, 10, or 5 mg resilin / g dry cell weight / hour; 5-40, 30, 20, or 10 mg resilin / g dry cell weight / hour; 10-40, 30, or 20 mg Recombinant resilin is produced at a rate of 20-40 mg resilin / g dry cell weight / hour or 30-40 mg resilin / g dry cell weight / hour. In other such embodiments, recombinant host cells are administered at concentrations exceeding 2 mg resilin / g dry cell weight / hour, 4 mg resilin / g dry cell weight / hour, 6 mg resilin / g dry cell weight / hour, 8 mg resilin / g dry cell weight / hour, 10 mg resilin / g dry cell weight / hour, 12 mg resilin / g dry cell weight / hour, 14 mg resilin / g dry cell weight / hour, 16 mg resilin / g dry cell weight / hour, 18 mg resilin / g dry cell weight / hour, 20 mg resilin / g dry cell weight / hour, 25 mg resilin / g dry cell weight / hour, or 30 mg resilin / g dry cell weight / hour; 2-40, 30, 20, 10, or 5 mg resilin / g dry cell weight / hour; 5-40, 30, 20, or 10 mg resilin / g dry cell weight / hour; 10-40, 30, or 20 mg Recombinant resilin is secreted at a rate of resilin / g dry cell weight / hour; 20-40 mg or 30 mg resilin / g dry cell weight / hour; or 30-40 mg resilin / g dry cell weight / hour.The identity of the recombinant resilin produced can be confirmed by HPLC quantification, Western blot analysis, polyacrylamide gel electrophoresis, and two-dimensional mass spectrometry (2D-MS / MS) sequence identification.

[0114] In some embodiments, recombinant host cells provided herein have a high secretory fraction of recombinant resilin provided herein. In some such embodiments, recombinant host cells have a secretory fraction of recombinant resilin, the secretory fraction being greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%; 50% to 100%, 90%, 80%, 70%, or 60%; 60% to 100%, 90%, 80%, or 70%; 70% to 100%, 90%, or 80%; 90% to 100%, or 90%; or 90% to 100%.

[0115] The production and secretion of recombinant resilin may be influenced by the number of copies of the secretory resilin coding sequence contained in the recombinant host cell and / or the rate of transcription of the secretory resilin coding sequence contained in the recombinant host cell. In some embodiments, the recombinant host cell contains one secretory resilin coding sequence. In other embodiments, the recombinant host cell contains two or more (e.g., three, four, five, or more) secretory resilin coding sequences. In some embodiments, the recombinant host cell contains a secretory resilin coding sequence functionally linked to a strong promoter. A non-limiting example of a strong promoter is the pGCW14 promoter of Pichia pastris. In some embodiments, the recombinant host cell contains a secretory resilin coding sequence functionally linked to a moderate promoter. A non-limiting example of such a moderate promoter is the pGAP promoter of Pichia pastris. In some embodiments, the recombinant host cell contains a coding sequence that encodes resilin under the regulation of a weak promoter.

[0116] The fermentation products provided herein include recombinant host cells as described herein and a culture medium suitable for growing recombinant host cells.

[0117] The fermented product is obtained by culturing recombinant host cells in a culture medium that provides nutrients required by recombinant host cells for cell survival and / or proliferation and for the secretion of recombinant resilin. Such culture media typically contain an excess of carbon sources. Non-limiting examples of suitable carbon sources include monosaccharides, disaccharides, polysaccharides, and combinations thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, xylose, arabinose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include raffinose, starch, glycogen, glycan, cellulose, chitin, and combinations thereof.

[0118] In some cases, the fermented product may be at least 1%, 5%, 10%, 20%, or 30% by weight of the total fermented product; 1% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%; 10% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%; 20% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, or 30%; Contains recombinant resilin in amounts of 30%-100%, 90%, 80%, 70%, 60%, 50%, or 40%; 40%-100%, 90%, 80%, 70%, 60%, or 50%; 50%-100%, 90%, 80%, 70%, or 60%; 60%-100%, 90%, 80%, or 70%; 70%-100%, 90%, or 80%; 80%-100% or 90%; or 90%-100%.

[0119] In some aspects, the fermented product contains at least 2g / L, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L, or 30g / L; 2g / L to 300g / L, 200g / L, 100g / L, 90g / L, 80g / L, 70g / L, 60g / L, 50g / L, 40g / L, 30g / L, 20g / L, or 10g / L; 10g / L to 300g / L, 200g / L, 100g / L, 90g / L, 80g / L, 70g / L, 60g / L, 50g / L, 40g / L, 30g / L, or 20g / L; 20g / L~300g / L, 200g / L, 100g / L, 90g / L , 80g / L, 70g / L, 60g / L, 50g / L, 40g / L, or 30g / L; 30g / L~300g / L, 200g / L, 100g / L, 90g / L, 80g / L, 70g / L, 60 g / L, 50g / L, or 40g / L; 40g / L to 300g / L, 200g / L, 100g / L, 90g / L, 80g / L, 70g / L, 60g / L, or 50g / L; 50g / L to 300g / L, 200g / L, 100g / L, 90g / L, 80g / L, 70g / L, or 60g / L; 60g / L to 300g / L, 200g / L, 100g / L, 90g / L, 80g / L, Alternatively, it contains 70g / L; 70g / L to 300g / L, 200g / L, 100g / L, 90g / L, or 80g / L; 80g / L to 300g / L, 200g / L, 100g / L, or 90g / L; 90g / L to 300g / L, 200g / L, or 100g / L; 100g / L to 300g / L or 200g / L; or 200g / L to 300g / L of recombinant resilience.

[0120] method A method for producing recombinant resilin described herein is further provided herein.

[0121] The aforementioned methods are generally carried out by conventional methods well known in the art, unless otherwise shown, as described in the various general and more specific references cited and described throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,1990;Taylor and Drickamer,Introduction to Glycobiology,Oxford Univ.Press,2003;Worthington Enzyme Manual,Worthington Biochemical Corp.,Freehold,NJ;Handbook of Biochemistry:Section A Proteins,Vol I,CRC Press,1976;Handbook of Biochemistry:Section A Proteins,Vol II,CRC Press,1976;Essentials of Glycobiology,Cold Spring Harbor Laboratory See Press, 1999.

[0122] In some embodiments, novel methods are used to secrete resilin extracellularly from host cells. In some embodiments, the method comprises the steps of constructing a vector containing a secretory resilin coding sequence (step 1001 in Figure 2), transforming the vector in host cells (step 1002 in Figure 2), and then culturing recombinant host cells to secrete resilin extracellularly (step 1003 in Figure 2). In some embodiments, the method is greater than 2 mg resilin / g dry cell weight / hour, 4 mg resilin / g dry cell weight / hour, 6 mg resilin / g dry cell weight / hour, 8 mg resilin / g dry cell weight / hour, 10 mg resilin / g dry cell weight / hour, 12 mg resilin / g dry cell weight / hour, 14 mg resilin / g dry cell weight / hour, 16 mg resilin / g dry cell weight / hour, 18 mg resilin / g dry cell weight / hour, 20 mg resilin / g dry cell weight / hour, 25 mg resilin / g dry cell weight / hour, or 30 mg resilin / g dry cell weight / hour; 2-40, 30, 20, 10, or 5 mg resilin / g dry cell weight / hour; 5-40, 30, 20, or 10 mg resilin / g dry cell weight / hour; 10-40, 30, or 20 mg The method comprises secreting resilin extracellularly at a rate of resilin / g dry cell weight / hour; 20-40 or 30 mg resilin / g dry cell weight / hour; or 30-40 mg resilin / g dry cell weight / hour. In some embodiments, the secreted resilin is then purified (step 1004 in Figure 2), and the purified resilin is crosslinked to form an elastomer (step 1005 in Figure 2). In some embodiments, the method provided herein comprises the step of transforming cells using the vector provided herein to obtain recombinant host cells provided herein (step 1002 in Figure 2). Methods for transforming cells using vectors are well known in the art.Non-limiting examples of such methods include calcium phosphate transfection, dendrimer transfection, liposome transfection (e.g., cationic liposome transfection), cationic polymer transfection, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, and viral transduction. Those skilled in the art may, based on knowledge in the art that certain techniques for introducing vectors work better for certain types of cells, select one or more methods suitable for transforming cells using the vectors provided herein.

[0123] In some embodiments, the method further comprises the step of culturing recombinant host cells provided herein in a culture medium under conditions suitable for obtaining the fermentates provided herein (step 1003 in Figure 2). In some embodiments, the conditions and culture medium are suitable for facilitating the secretion of recombinant proteins from the recombinant host cells into the culture medium. Culture media suitable for use in these methods are known in the art, as are suitable culture conditions. Exemplary details of the culture of yeast host cells are described in Idiris et al., Appl. Microbiol. Biotechnol. 86:403-417, 2010; Zhang et al., Biotechnol. Bioprocess. Eng. 5:275-287, 2000; Zhu, Biotechnol. Adv. 30:1158-1170, 2012; Li et al., MAbs 2:466-477, 2010.

[0124] In some embodiments, the method further comprises the step of purifying secreted recombinant resilin from a fermentation provided herein (step 1004 in Figure 2) in order to obtain recombinant resilin provided herein. Purification can be carried out by a variety of methods known in the art for purifying secreted proteins from fermentations. Common steps in such methods include centrifugation (to remove cells), followed by precipitation of the protein using a precipitating agent or other suitable cosmotropes (e.g., ammonium sulfate). The precipitated protein can then be separated from the supernatant by centrifugation and resuspended in a solvent (e.g., phosphate-buffered saline [PBS]). The suspended protein can be dialyzed to remove dissolved salts. Furthermore, the dialyzed protein can be heated to denature other proteins, and the denatured proteins can be removed by centrifugation. Optionally, the purified recombinant resilin can be coacervated.

[0125] In various embodiments, methods for purifying secretory recombinant proteins from fermentation products may include various centrifugation steps, along with a step of solubilizing the proteins in the whole cell broth or cell pellet with a known chaotrope such as urea or guanidine thiocyanate.

[0126] In some embodiments, the methods provided herein further include the step of crosslinking recombinant resilin to obtain the recombinant resilin composition provided herein (step 1005 in Figure 2). Methods for crosslinking proteins are known in the art. In some embodiments, crosslinking is achieved via enzymatic crosslinking (e.g., using horseradish peroxidase). In other embodiments, crosslinking is achieved via photochemical crosslinking (see, for example, Elvin CM, Carr AG, Huson MG, Maxwell JM, Pearson RD, Vuocolo T, Liyou NE, Wong DCC, Merritt DJ, Dixon NE. Nature 2005, 437, 999-1002; Whittaker JL, Dutta NK, Elvin CM, Choudhury NR. Journal of Materials Chemistry B 2015, 3, 6576-79; Degtyar E, Mlynarczyk B, Fratzl P, Harrington MJ. Polymer 2015, 69, 255-63). In some embodiments, crosslinking is achieved via chemical crosslinking (see, for example, Renner JN, Cherry KM, Su RSC, Liu JC. Biomacromolecules 2012, 13, 3678-85; Charanti, MB, Ifkovits, JL, Burdick, JA, Linhardt JG, Kiick, KL. Soft Matter 2009, 5, 3412-16; Li LQ, Tong ZX, Jia XQ, Kiick KL. Soft Matter 2013, 9, 665-73; Li L, Mahara A, Tong Z, Levenson EA, McGann CL, Jia X, Yamaoka T, Kiick KL. Advanced Healthcare Materials 2016, 5, 266-75). In some embodiments, crosslinking is achieved via tyrosine residues. In other embodiments, crosslinking is achieved via lysine residues. In some embodiments, crosslinking is achieved via cysteine ​​residues.In some embodiments, crosslinking is performed using transglutaminase (see, for example, Kim Y, Gill EE, Liu JC. Enzymatic Cross-Linking of Resilin-Based Proteins for Vascular Tissue Engineering Applications. Biomacromolecules. 17(8):2530-9). In some embodiments, crosslinking is performed using poly(ethylene glycol) (PEG) (McGann CL, Levenson EA, Kiick KL. Macromol. Chem. Phys. 2013, 214, 203-13; McGann CL, Akins RE, Kiick KL. Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure. Biomacromolecules. 2016; 17(1):128-40). In some embodiments, crosslinking is performed in a container or mold so that the resulting recombinant resilin composition has a specific shape or form. [Examples]

[0127] Example 1: Generation of Pichia pastris recombinant host cells that secrete recombinant resilin Recombinant host cells of Pichia pastris (Comagatella faffii) that secrete recombinant resilin were generated by transforming the HIS+ derivative of GS115 (NRRL Y15851) Pichia pastris (Comagatella faffii) with a vector containing a secreted resilin coding sequence.

[0128] Each of the vectors contained three regirin coding sequences fused in-frame with an N-terminal secretory signal (α-conjugation factor reader sequence and pro sequence), and in some cases contained a C-terminal 3×FLAG tag (SEQ ID NO: 45) (see Figure 3). Each secretory regirin coding sequence was flanked by a promoter (pGCW14) and a terminator (tAOX1 pA signal). The vectors further contained a targeting region that could direct the integration of the three secretory regirin coding sequences into the HSP82 locus of the Pichia pastris genome, a dominant resistance marker for the selection of bacterial and yeast transformants, and a bacterial replication origin.

[0129] Regirin coding sequences were obtained from scientific literature and public sequence database searches. Nucleotide sequences were translated into amino acid sequences and then codon-optimized. Both full-length and truncated regirin sequences were selected. The selected secreted regirin coding sequences are listed in Table 1.

[0130] (Table 1) Exemplary full-length and truncated resilin amino acid sequences and recombinant host strains TIFF0007856997000004.tif177161

[0131] To generate host strains containing three copies of each incorporated secretory regirin coding sequence, the vectors were transformed in Pichia pastris using electroporation. The transformants were seeded onto YPD agar plates supplemented with antibiotics and incubated at 30°C for 48 hours.

[0132] Clones derived from each final transformation were inoculated into 400 μL of buffered glycerol complex medium (BMGY) in a 96-well block and incubated at 30°C for 24 hours with stirring at 1,000 rpm. Samples were removed, recombinant host cells were pelleted by centrifugation, and the supernatant was collected and performed on an SDS-PAGE gel for analysis of resilin content via Coomasiegel and Western blotting (for polypeptides containing 3×FLAG tags). For FLAG-tagged proteins, the remaining culture was used to inoculate minimal medium cultures in duplicate for ELISA analysis. One duplicate was pelleted and the supernatant was measured directly. The second duplicate was extracted with guanidine thiocyanate, and both intracellular and extracellular fractions were measured.

[0133] As shown in Figures 4B and 4C, recombinant regirins from numerous species were successfully expressed in Pichia pastris recombinant host cells (Note: Some proteins have very few basic residues and are therefore difficult to detect by Coomassi, but they do produce a signal in Western blot). As shown in Figure 4A, the recombinant host cells secreted up to 90% of the recombinant regirins produced.

[0134] Example 2: Measurement of production levels of recombinant regirin in Pichia pastris recombinant host cells that express and secrete recombinant regirin. To measure production, three clones of each recombinant host cell were inoculated into 400 μL of BMGY in a 96-well square-well block and incubated at 30°C for 48 hours with stirring at 1,000 rpm. After 48 hours of incubation, 4 μL of each culture was used to inoculate into 400 μL of minimal medium in a 96-well square-well block, and then incubated at 30°C for 48 hours with stirring at 1,000 rpm. 400 μL of 5 M guanidine thiocyanate was added to the culture, and the mixture was pelleted by centrifugation. The supernatant was saved, and the pellet was resuspended in 800 μL of 2.5 M guanidine thiocyanate. The resuspended cells were physically lysed using beads, and the lysed cell mixture was pelleted by centrifugation, and the supernatant was saved. The resilin concentration of each fraction was determined by direct enzyme-linked immunosorbent assay (ELISA) analysis to quantify the 3×FLAG epitope (Figures 5A and 5B).

[0135] Example 3: Purification of recombinant resilin Non-FLAG-tagged Ds_ACB and Ae_A polypeptides were selected for purification and crosslinking. RMs1221 (expressing Ds_ACB) and RMs1224 (expressing Ae_A) strains were grown in 500 mL of BMGY in flasks at 30°C for 48 hours with stirring at 300 rpm.

[0136] The purification protocol was adapted from Lyons et al. (2007). Cells were pelleted by centrifugation, and the supernatant was collected. Proteins were precipitated by the addition of ammonium sulfate. The precipitated proteins were resuspended in a small amount of phosphate-buffered saline (PBS), and the resuspended sample was dialyzed against PBS to remove salts. The dialyzed sample was then heated to denature the native proteins, and the denatured proteins were removed by centrifugation. The retained supernatant contained purified resilin polypeptides. Optionally, the retained supernatant was cooled to induce coacervation, resulting in a concentrated lower phase and a dilute upper phase.

[0137] As shown in Figure 6, Ae_A was obtained in a relatively pure form, while Ds_ACB produced three bands at 70kDa, 50kDa, and 25kDa.

[0138] Example 4: Crosslinking of purified and secreted recombinant resilience The concentrated Ds_ACB resilin was crosslinked via one of two methods: photocrosslinking (adapted from Elvin et al. 2005) and enzymatic crosslinking (adapted from Qin et al. 2009).

[0139] For photocrosslinking, resilin proteins were mixed with ammonium persulfate and tris(bipyridine)ruthenium(II) ([Ru(bpy)3]2+). When the mixture was exposed to bright white light, it subsequently formed a rubbery solid.

[0140] For enzymatic crosslinking, resilin proteins were mixed with horseradish peroxidase (HRP) and hydrogen peroxide. When the mixture was incubated at 37°C, it subsequently formed a rubbery solid.

[0141] Example 5: Preparation of recombinant resilin blocks The RMs1221 strain (expressing Ds_ACB resilin) ​​was run in two 2L fermenters to produce larger quantities of protein.

[0142] The strains were grown in a minimal basal salt medium containing 15 g / L glucose and 1 g / L L81 defoamer as starting feed, in a stirred fermentation vessel adjusted to 30°C with an airflow of 1 VVM and minimal stirring at 700 rpm. The pH of the ferment was adjusted to 5 by the on-demand addition of ammonium hydroxide. When the batch glucose was depleted, glucose was added via a programmed feed recipe designed to maintain an oxygen uptake rate of 120 mmol / L / hour, the temperature was reduced to 25°C, and the dissolved oxygen was maintained at 20%. The ferment was collected after 70 hours at a cell density of approximately 700–800 OD.

[0143] The aforementioned protein was purified as described in Example 3 and combined with the reagent for enzymatic crosslinking as described in Example 4. The crosslinking mixture was filled into small cylindrical, rectangular, spherical, and shoe-shaped molds and finally incubated at 37°C. The resulting recombinant resilin solid is shown in Figure 7.

[0144] Example 6: Material testing of resilin solid The resilin cylinder prepared as described in Example 5 was subjected to a compression test using a rheometer. The recombinant resilin cylinder was able to be compressed from an initial height of 7.3 mm (average width 5.4 mm) to less than 0.66 mm without fracture. As shown in Figure 8, the cylinder returned to a height of 6.7 mm (average width 5.6 mm) upon release of the compression load.

[0145] Example 7: Method for recovering full-length recombinant resilin from the entire cell broth In accordance with Example 1 described above, various recovery and separation techniques were used to purify Ds_ACB (SEQ ID NO:1) produced in strains with a 3×FLAG tag (RMs1209) and strains without a 3×FLAG tag (RMs1221).

[0146] The first sample set was prepared by centrifuging the entire cell broth to produce a first cell pellet and first supernatant, and then extracting the first supernatant to produce a clear cell broth. The first supernatant was then precipitated using ammonium sulfate and centrifuged to produce a second pellet and second supernatant, and the second supernatant was discarded. The second pellet was then resuspended in PBS for dialysis. The dialyzed solution was then subjected to high temperature to denature proteins other than Ds_ACB, which are stable at high temperatures. The dialyzed denatured solution was centrifuged to remove the denatured proteins and produce a third pellet and third supernatant. The third supernatant from the denatured solution was retained and then coacervated by cooling the third supernatant to induce phase separation into a dense lower layer and an upper layer containing Ds_ACB. These samples are referred to as the “CCB” sample in Table 2 below and herein. In some CCB samples, multiple coacervations were performed by incubating the lower layer at low temperatures to preserve the lower layer and induce further phase separation. These CCB samples are referred to as the “first coacervation” sample and the “second coacervation” sample in Table 2 below and in this specification, respectively.

[0147] A second sample set was prepared by centrifuging the entire cell broth to produce a first pellet of cells and proteins proximal to the cells (e.g., adhering to cells, on the cell surface) and / or insoluble proteins (e.g., protein aggregates), and a first supernatant, and then discarding the first supernatant to obtain the first pellet. The first pellet was resuspended in guanidine thiocyanate to solubilize Ds_ACB. The resuspended material was centrifuged again to produce a second pellet and a second supernatant. The second supernatant was then dialyzed against PBS and subjected to high temperature to denature proteins other than Ds_ACB, and centrifuged to produce a third pellet and a third supernatant. The third supernatant was subjected to coacervation by cooling to give phase separation into a dense lower layer and an upper layer containing Ds_ACB. These samples are referred to as “gel layer” samples in Table 2 below and herein. In some gel layer samples, multiple coacervations were performed by incubating the underlying layer at a lower temperature to retain the underlying layer and induce further phase separation. These gel layer samples are referred to as “first coacervation” samples and “second coacervation” samples in Table 2 below and herein.

[0148] A third sample set was prepared by centrifuging the entire cell broth to produce a pellet and supernatant, then discarding the supernatant to obtain a pellet of cells and proteins proximal to the cells (e.g., adhering to the cells, on the cell surface) and / or insoluble proteins (e.g., protein aggregates). To solubilize the proteins proximal to the cells, the cell pellet was resuspended in guanidine thiocyanate. The resuspended mixture was again centrifuged to produce a second cell pellet and a second supernatant. The second supernatant was then precipitated with ammonium sulfate and centrifuged to produce a third pellet and a third supernatant. The third pellet was suspended in guanidine thiocyanate, then dialyzed against PBS and subjected to high temperature to denature proteins other than Ds_ACB, and centrifuged to produce a fourth supernatant and a fourth pellet. The fourth supernatant was then subjected to coacervation by cooling to give phase separation. These samples are referred to as “precipitated gel layer” samples in Table 2 below and herein.

[0149] A single sample was prepared by adding urea to the entire cell broth to solubilize the proteins, and then centrifugating the entire cell broth to produce a first pellet and a first supernatant. The first supernatant was then precipitated using ammonium sulfate and centrifuged to produce a second pellet and a second supernatant. The second supernatant was discarded, and the second pellet was then resuspended in guanidine thiocyanate, dialyzed against PBS, and then subjected to high temperature to denature proteins other than Ds_ACB, and centrifuged again to produce a third pellet and a third supernatant. The third supernatant was then coacervated by cooling to induce phase separation into a dense lower layer and an upper layer containing Ds_ACB. This sample is referred to as the “urea WCBE” sample in Table 2 below and herein.

[0150] Another single sample was prepared by centrifuging the entire cell broth to produce a first pellet and a first supernatant, and then discarding the first supernatant to obtain a first pellet of cells and proteins proximal to the cells (e.g., adherent to cells, on the cell surface) and / or insoluble proteins (e.g., protein aggregates). The first pellet of cells was resuspended in guanidine thiocyanate to solubilize the proteins. The resuspended mixture was centrifuged again to produce a second pellet of cells and a second supernatant. The second supernatant was then dialyzed against PBS and then centrifuged to produce a film separating the heavy phase of proteins, the light phase of the supernatant, and the heavy phase from the light phase. The heavy phase of proteins was then isolated by discarding the light phase and the film. This sample is referred to as the “high-density layer” sample in Table 2 below and herein.

[0151] Table 2 (below) lists various combinations of strains and recovery techniques, along with the relative amounts of degradation observed in the gels shown in Figure 9. As shown in Figure 9, samples E, F, G, K, and L showed a band at approximately 110 kDa and minimal or weak bands at lower molecular weights (labeled "minimal" in Table 2). Samples A, B, C, D, G, I, and J had degradation products corresponding to bands at approximately 90 kDa, 30 kDa, 22 kDa, 17 kDa, and 12 kDa (labeled "substantial" in Table 2). Of these, samples A and I also showed a band at approximately 110 kDa, indicating the presence of full-length resilin. Thus, the "gel layer" samples produced full-length resilin, while the CCB samples produced degradation products, sometimes in addition to full-length resilin (e.g., sample A) or without full-length resilin (e.g., samples C and D). The urea WCBE samples produced only degradation products. The CCB / precipitated gel layer shows a combination of materials isolated from both the CCB purification method and the gel layer purification method.

[0152] (Table 2) Samples from recovery methods yielding full-length resilin and degradation products TIFF0007856997000005.tif87160

[0153] To confirm that the 110 kDa bands shown in samples A, I, E, F, G, K, and L correspond to full-length resilin (SEQ ID NO: 1), the 110 kDa band from sample H (indicated by the arrow in Figure 9) was excised and sent for N-terminal sequencing by Edman degradation. Edman degradation is a periodic method in which amino acid residues are cleaved one by one and identified by chromatography. There are three steps in this periodic method. In step 1, the PITC reagent is coupled to the N-terminal amino group under alkaline conditions. In step 2, the N-terminal residue is cleaved in an acidic medium. In step 3, the residue coupled with PITC is transferred to a flask, converted to a PTH residue, and identified by HPLC chromatography. The next cycle is then started for the identification of the next N-terminal residue. Edman degradation analysis was performed on a Shimadazu PPSQ-33 sequencer and PVDF membrane.

[0154] Figure 10 shows the full-length Seychelles elegirin sequence (Ds_ACB) expressed along with signal sequences that are later cleaved. The first sequence (italicized) is the α-conjugation precursor protein signal sequence (SEQ ID NO: 46), which is cleaved twice after transcription by a signal peptidase and then cleaved by Kex2. The second sequence (bold) is the EAEA repeat (SEQ ID NO: 47), which is cleaved by Ste13. The third sequence (lowercase) corresponds to the full-length Seychelles elegirin (SEQ ID NO: 1). The fourth sequence (bold italicized) corresponds to the linker sequence (SEQ ID NO: 46). The fifth sequence (underlined) corresponds to the 3×FLAG tag (SEQ ID NO: 45).

[0155] Edman sequencing confirmed that the N-terminus of the protein sequence in the approximately 110 kDa band corresponds to the full-length Seychelles elegiline sequence. Specifically, N-terminal sequencing showed that the N-terminus corresponds to either the full-length Seychelles elegiline sequence "EAEA" containing the EAEA repeat or the full-length Seychelles elegiline sequence "GRPE" without the EAEA repeat.

[0156] Example 8: Quantification of the stability of crosslinked resilin Resilin samples produced by the method described in Example 7, containing fluctuating levels of degradation products and full-length resilin, were subjected to enzymatic crosslinking as described in Example 4. The stability of the crosslinked samples was assessed over time by determining the duration for which each crosslinked sample remained solid through daily observation. Table 3 shows the duration of solidity for each crosslinked sample. As shown in Table 3, samples containing full-length resilin had a longer stability duration than samples without full-length resilin.

[0157] (Table 3) Stability of crosslinked resilin TIFF0007856997000006.tif73128

[0158] Additional considerations The above description of the embodiments of this disclosure is provided for illustrative purposes only; it is not exhaustive and is not intended to limit the claims to the exact embodiments disclosed. Those skilled in the art will understand that many modifications and variations are possible in light of the above disclosure.

[0159] The language used herein has been chosen primarily for readability and teaching purposes and not to indicate or limit the boundaries of the subject matter of the invention. Accordingly, the scope of this disclosure is limited not by this detailed description but by the claims issued relating to applications based on this disclosure. Accordingly, the disclosure of embodiments is illustrative and not intended to limit the scope of the invention as shown in the claims.

[0160] List of sequences TIFF0007856997000007.tif206155TIFF0007856997000008.tif187155TIFF000 7856997000009.tif218155TIFF0007856997000010.tif183155TIFF00078569970 00011.tif222155TIFF0007856997000012.tif207155TIFF0007856997000013.t if176155TIFF0007856997000014.tif222155TIFF0007856997000015.tif126155

Claims

1. A step of culturing a population of recombinant host cells in a ferment, wherein the recombinant host cells contain a vector comprising a secreted resilin coding sequence, the recombinant host cells secrete recombinant resilin protein encoded by the secreted resilin coding sequence, the recombinant host cells are methylotrope yeast cells, and the recombinant methylotrope yeast host cells produce a secreted fraction of the recombinant resilin, the secreted fraction being more than 50% of the total recombinant resilin protein expressed by the recombinant host cells; and A step of purifying the recombinant resilin protein from the fermented product. A method for preparing a composition containing recombinant resilin protein, including the above.

2. The method according to claim 1, wherein the recombinant resilin protein is a full-length or truncated native resilin.

3. The aforementioned native resilin is found in the following insects: Drosophila sechellia, Acromyrmex echinatior, Aeshna, Haematobia irritans, Ctenocephalides felis, Bombus terrestris, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Pediculus humanus corporis, Anopheles gambiae, Glossina morsitans, Atta cephalotes, Anopheles darlingi, and Acyrthosiphon The method according to claim 1, wherein the organism is derived from an organism selected from the group consisting of pisum), black fruit fly (Drosophila virilis), Kirishima fungus fruit fly (Drosophila erecta), sand fly (Lutzomyia longipalpis), giant assassin bug (Rhodnius prolixus), red imported fire ant (Solenopsis invicta), tropical house mosquito (Culex quinquefasciatus), melon fly (Bactrocera cucurbitae), and Trichogramma pretiosum.

4. The method according to claim 1, wherein the recombinant resilin protein comprises SEQ ID NO:

1.

5. The method according to claim 1, wherein the recombinant resilin protein comprises SEQ ID NO:

4.

6. The method according to claim 1, wherein the recombinant resilin protein includes an α-conjugation factor secretion signal.

7. The method according to claim 1, wherein the recombinant resilin protein includes a FLAG tag.

8. The method according to claim 1, wherein the vector comprises a plurality of secreted resilin coding sequences.

9. The method according to claim 1, wherein the recombinant methanolyzool host cell is a species selected from the group consisting of Pichia (Komagataella phaffii) pastoris, Hansenula polymorpha, Pichia (Scheffersomyces) stipitis, Pichia methanolica, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida pseudotropicalis, Candida utilis, and Candida versatilis.

10. The method according to claim 1, wherein the recombinant methylotrope yeast host cells produce the recombinant resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour.

11. The method according to claim 1, wherein the recombinant methylotrope yeast host cells secrete the recombinant resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour.

12. The method according to claim 1, wherein more than 80% of the recombinant resilin is present outside the recombinant methylotrope yeast host cells in the ferment.

13. The method according to claim 1, wherein the fermented product contains at least 2 g of recombinant resilin per liter.

14. The step of purifying the recombinant resilin protein is To produce a first pellet fraction and a first supernatant fraction by centrifuging the fermented product; and Isolating the recombinant resilin protein from the first pellet fraction. The method according to claim 1, including the method described in claim 1.

15. The step of purifying the recombinant resilin protein is To produce a solution in which the recombinant resilin protein is soluble, chaotrope is added to the first pellet fraction; To produce a second supernatant fraction and a second pellet fraction by centrifuging the first pellet fraction containing the chaotrope; and To isolate soluble full-length resilin from the second supernatant fraction. The method according to claim 14, further comprising:

16. A vector for methylotrope yeast cells comprising multiple secreted resilin coding sequences, wherein the secreted resilin coding sequences encode polypeptides containing α-conjugation factor secretion signals.

17. The vector according to claim 16, wherein the secreted resilin coding sequence codes for a full-length or truncated native resilin.

18. The vector according to claim 16, wherein the secreted resilin coding sequence codes for a modified full-length or truncated native resilin.

19. The vector according to claim 18, wherein the modified resilin comprises the addition of an amino acid residue, the deletion of an amino acid residue, the substitution of an amino acid residue, or the repositioning of an amino acid residue, and the amino acid residue can be crosslinked with another resilin.

20. The vector according to any one of claims 17 to 19, wherein the full-length or shortened native resilin is derived from an organism selected from the group consisting of Drosophila melanogaster, Leafcutter ants, Aeshna genus, Stingray flies, Cat fleas, Bombus ignitus, Confused flour bees, Honeybees, Parasitic wasps, Body lice, Anopheles gambia, Glossina moorcitans, Atta cephalotes, Anopheles darlingzi, Pea aphids, Drosophila melanogaster, Drosophila kirishimaensis, Sand flies, Assassin bugs, Red imported fire ants, Culex pipiens, Culex cephalospermum, Trichogramma plethiosum.

21. The vector according to claim 20, wherein the secreted resilin-coding sequence encodes a polypeptide containing SEQ ID NO:

1.

22. The vector according to claim 20, wherein the secreted resilin-coding sequence encodes a polypeptide containing SEQ ID NO:

4.

23. The vector according to claim 16, wherein the secreted resilin coding sequence codes for recombinant resilin comprising one or more A repeats or quasi-A repeats.

24. The vector according to claim 16, wherein the secreted resilin coding sequence codes for recombinant resilin comprising one or more B repeats or quasi-B repeats.

25. The vector according to claim 16, wherein the secreted resilin coding sequence codes for recombinant resilin, and the recombinant resilin comprises only one or more A repeats or quasi-A repeats, or one or more B repeats or quasi-B repeats.

26. The vector according to claim 16, wherein the secreted resilin coding sequence codes for recombinant resilin comprising one or more A repeats or quasi-A repeats and one or more B repeats or quasi-B repeats.

27. The vector according to any one of claims 23 to 26, further comprising a chitin-binding domain, wherein the recombinant resilin.

28. The vector according to any one of claims 16 to 27, wherein the secreted resilin coding sequence includes a FLAG tag.

29. The vector according to claim 16, comprising three secretory resilin coding sequences.

30. The vector according to any one of claims 16 to 29, wherein the secreted resilin coding sequence is functionally linked to a constructive or inducible promoter.

31. A recombinant host cell comprising one or more vectors according to any one of claims 16 to 30, wherein the recombinant host cell is a methylotrope yeast cell.

32. Recombinant host cell according to claim 31, the species is selected from the group consisting of Pichia (Comagatella faffii) pastris, Hanzenula polymorpha, Pichia (Scheffersomyces) stipitis, Pichia metanorica, Candida echelsii, Candida gilliermondi, Candida humilis, Candida liporitica, Candida pseudotropicalis, Torula yeast, and Candida versatilis.

33. A recombinant host cell according to claim 31, comprising three vectors according to any one of claims 16 to 30.

34. Recombinant host cells according to any one of claims 31 to 33, which produce recombinant resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour.

35. The recombinant host cell according to any one of claims 31 to 34, wherein the recombinant methylotrope yeast host cell produces a secreted fraction of recombinant resilin, and the secreted fraction is more than 50% of the total recombinant resilin protein expressed by the recombinant host cell.

36. Recombinant host cells according to any one of claims 31 to 35, which secrete resilin at a rate exceeding 2 mg resilin / g dry cell weight / hour.

37. A fermentation comprising recombinant methylotrope yeast host cells according to any one of claims 31 to 36 and a culture medium suitable for growing the recombinant host cells.

38. The fermented product according to claim 37, comprising at least 2 g of recombinant resilin per liter.

39. The fermented product according to claim 37, wherein more than 80% of recombinant resilin is present outside the recombinant methylotrope yeast host cells.

40. The fermented product according to any one of claims 37 to 39, wherein the recombinant resilin is full-length recombinant resilin.

41. A method for preparing a composition containing recombinant resilin, comprising the step of culturing recombinant methylotrope yeast host cells according to any one of claims 31 to 36 in order to produce a ferment under conditions that promote the secretion of recombinant resilin.

42. The method according to claim 41, further comprising the step of purifying the recombinant resilin to produce full-length native resilin.

43. The process of purifying the recombinant resilin in order to produce full-length native resilin is To produce a first pellet fraction and a first supernatant fraction by centrifuging the fermented product; and To isolate recombinant resilin protein from the first pellet fraction. The method according to claim 42, including the method described in claim 42.

44. Isolating the recombinant resilin protein from the first pellet fraction is To produce a solution in which the recombinant resilin protein is soluble, chaotrope is added to the first pellet fraction; To produce a second supernatant fraction and a second pellet fraction by centrifuging the first pellet fraction containing the chaotrope; and Isolating the recombinant resilin protein from the second supernatant fraction. The method according to claim 43, including the method described in claim 43.

45. The method according to any one of claims 41 to 44, further comprising the step of crosslinking a plurality of the recombinant resilin.

46. The method according to claim 45, wherein the crosslinking is enzymatic crosslinking.

47. The method according to claim 45, wherein the crosslinking is photochemical crosslinking.

48. The method according to any one of claims 41 to 47, wherein the recombinant resilin protein comprises a full-length resilin protein.

49. A fermentation product comprising culture medium and recombinant methylotrope yeast host cells, The ferment comprising recombinant methylotrope yeast host cells comprising a vector, the vector comprising a secretory resilin coding sequence, and the recombinant host cells secreting recombinant resilin at a rate of at least 2 mg / g dry cell weight / hour.

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