Cross-linked elastomeric proteins in polar nonaqueous solvents and uses thereof

Abstract

To provide new methods of cross-linking recombinant resilin proteins that do not leave behind degradation-causing impurities and can be performed efficiently on a large scale, compositions comprising the cross-linked recombinant resilin, and methods of producing the compositions.SOLUTION: A method of cross-linking recombinant resilin comprises the steps for: providing a composition comprising purified recombinant resilin; placing the recombinant resilin in a cross-linking solution comprising ammonium persulfate; and incubating the recombinant resilin in the cross-linking solution at a temperature of at least 60°C, thereby generating a cross-linked recombinant resilin solid composition. The cross-linking solution may be free of a photocatalyst or a cross-linking enzyme.SELECTED DRAWING: Figure 12

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 700,197, filed July 18, 2018, the contents of which are incorporated by reference in their entirety. [Background technology]

[0002] background Elastomeric proteins are polypeptides that exhibit viscoelastic mechanical properties and include elastin, resilin, abductin, and octopus arterial elastomers. Resilin is a particularly interesting elastomeric protein because it consumes very little energy during loading and unloading. Resilin is found in many insects, and the low energy consumption allows many insect species the special ability to jump or spin their wings very efficiently. Due to its unique properties, resilin is an interesting elastomeric material that may have many industrial applications. Therefore, what is needed are new resilin compositions and methods of making them that have desirable mechanical properties and are suitable for large-scale, efficient manufacturing.

[0003] Various variants of native resilin and resilin-like proteins (based on the resilin sequence) have been recombinantly produced by numerous groups in E. coli cultures and isolated by lysing the cells to extract the recombinantly expressed proteins and using affinity chromatography techniques to purify them (Elvin et al., 2005; Charati et al., 2009; McGann et al., 2013). Recombinantly produced resilin and resilin-like proteins have been cross-linked (see, e.g., Elvin et al., 2005; Qin et al., 2011) targeting tyrosine residues that also form cross-links in native resilin. Recombinantly produced resilin has also been cross-linked targeting lysine residues (Li et al., 2011) or cysteine ​​residues (McGann et al., 2013). Cross-linking methods include enzymatically catalyzed cross-linking and photoactivated cross-linking. Enzymes for enzymatic catalysis of crosslinking can be difficult to remove from the final composition, while photoactivated crosslinking can be inefficient because only the surface is exposed to light, while also leaving behind impurities. Enzyme impurities can degrade resilin, while impurities from either the enzyme or photocatalysis that remain in the final solid crosslinked composition can result in undesirable mechanical properties. Therefore, what is needed is a new method of crosslinking recombinant resilin protein that does not leave behind impurities that cause degradation and that can be performed efficiently on a large scale. Summary of the Invention

[0004] Disclosed herein are improved crosslinked resilin compositions and methods for making these improved compositions, including a new method for crosslinking the resilin compositions and the use of polar non-aqueous solvents to tailor the material properties of the crosslinked resilin solid compositions.

[0005] In some embodiments, provided herein is a method for cross-linking recombinant resilin, the method comprising the steps of providing a composition comprising purified recombinant resilin, placing the recombinant resilin in a cross-linking solution comprising ammonium persulfate, and incubating the recombinant resilin in the cross-linking solution at a temperature of at least 60°C, thereby producing a cross-linked recombinant resilin solid composition.

[0006] In some embodiments, the incubation is carried out for at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, or at least 2 hours.

[0007] In some embodiments, the recombinant resilin in the crosslinking solution is incubated at a temperature between 60°C and 85°C, between 70°C and 85°C, or between 75°C and 85°C.

[0008] In some embodiments, the incubation is carried out for at least 2 hours.

[0009] In some embodiments, the cross-linking solution does not include a photocatalyst or a cross-linking enzyme.

[0010] In some embodiments, the crosslinked recombinant resilin solid composition is stable at room temperature for more than 5 days, more than 10 days, more than 20 days, or more than 40 days.

[0011] In some embodiments, purified recombinant resilin is prepared by recombinantly expressing a gene encoding the recombinant resilin in a modified organism in culture and purifying the expressed recombinant resilin from the culture.

[0012] Also provided herein, according to some embodiments, is a composition comprising a cross-linked recombinant resilin, the recombinant resilin being cross-linked by exposing the resilin to ammonium persulfate and heat. In some embodiments, the composition does not include a cross-linking enzyme. In some embodiments, the composition does not include a photocatalyst.

[0013] In some embodiments, provided herein is a recombinant resilin composition comprising a cross-linked recombinant resilin in a polar non-aqueous solvent.

[0014] In some embodiments, the polar non-aqueous solvent is a protic solvent. In some embodiments, the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol. In some embodiments, the polar non-aqueous solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DMSO.

[0015] In some embodiments, the composition comprises 20-40% by weight resilin.

[0016] In some embodiments, the resilin is at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a fraction of total resilin as measured by size exclusion chromatography.

[0017] In some embodiments, the polar non-aqueous solvent is at least 60% by volume, at least 70% by volume, at least 80% by volume, at least 90% by volume, at least 95% by volume, at least 98% by volume, or at least 99% by volume of the solvent.

[0018] In some embodiments, the recombinant resilin composition has a greater elastic modulus than an analogous cross-linked recombinant resilin in an aqueous solvent.

[0019] In some embodiments, the recombinant resilin composition comprises a hardness of at least 10, as measured using a Shore OO durometer according to ASTM D2240.

[0020] In some embodiments, the recombinant resilin composition comprises a hardness of about 10 to about 50, as measured using a Shore 00 durometer according to ASTM D2240.

[0021] In some embodiments, the recombinant resilin composition comprises a rebound resilience of about 40% to about 60% as measured by ASTM D7121.

[0022] In some embodiments, the recombinant resilin composition comprises a compressive stress at 25% of about 6 to about 8 psi as measured by ASTM D575.

[0023] In some embodiments, the recombinant resilin composition does not undergo an elastic to plastic transition at a compressive force of less than 2 kN as measured by the Zwick compression test, hi some embodiments, the recombinant resilin composition is a foam material.

[0024] Also provided herein, according to some embodiments, is a method of preparing a recombinant resilin solid, the method comprising providing a crosslinked recombinant resilin solid composition in an aqueous solvent and exchanging the aqueous solvent with a polar non-aqueous solvent.

[0025] In some embodiments, the polar non-aqueous solvent is a protic solvent. In some embodiments, the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol. In some embodiments, the polar non-aqueous solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DMSO.

[0026] In some embodiments, the solvent exchange is carried out for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours.

[0027] In some embodiments, the solvent exchange is carried out at about 60°C.

[0028] In some embodiments, the crosslinked recombinant resilin solid composition comprises at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a fraction of total resilin as measured by size exclusion chromatography.

[0029] In some embodiments, the crosslinked recombinant solid compositions are prepared by the ammonium persulfate and heating methods described herein.

[0030] Also provided herein, according to some embodiments, is a method of tailoring material properties of a solid composition comprising a crosslinked recombinant resilin solid, the method comprising providing a crosslinked recombinant resilin solid composition comprising an aqueous solvent, and performing a solvent exchange to replace the aqueous solvent with a polar non-aqueous solvent, in some embodiments, replacing the solvent alters the elastic modulus, elasticity, hardness, maximum elastic compressive load, or material lifetime of the crosslinked recombinant resilin solid composition.

[0031] Also provided herein, according to some embodiments, is a method of preparing a recombinant crosslinked resilin solid, the method comprising the steps of providing a composition comprising purified recombinant resilin, placing the recombinant resilin in an aqueous solvent comprising ammonium persulfate, incubating the recombinant resilin in the aqueous solvent at a temperature of at least 60°C, thereby producing a crosslinked recombinant resilin solid composition, and exchanging the aqueous solvent with a polar non-aqueous solvent selected from the group consisting of glycerol, propylene glycol, ethylene glycol, and DMSO.

[0032] Also provided herein, according to some embodiments, is a method of preparing a recombinant resilin foam, the method comprising providing a cross-linked recombinant resilin solid composition in an aqueous solvent, exchanging the aqueous solvent with a polar non-aqueous solvent, and introducing one or more gas bubbles into the cross-linked recombinant resilin solid composition.

[0033] In some embodiments, the polar non-aqueous solvent is a protic solvent. In some embodiments, the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol. In some embodiments, the polar non-aqueous solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DMSO.

[0034] In some embodiments, the solvent exchange is carried out for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours.

[0035] In some embodiments, the solvent exchange is carried out at about 60°C.

[0036] In some embodiments, the crosslinked recombinant resilin solid composition comprises at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a fraction of total resilin as measured by size exclusion chromatography.

[0037] In some embodiments, the crosslinked recombinant solid compositions are prepared by the ammonium persulfate and heating methods described herein.

[0038] In some embodiments, the introducing comprises adding a foaming agent to the cross-linked recombinant resilin solid composition.

[0039] In some embodiments, the blowing agent comprises a chemical blowing agent. In some embodiments, the chemical blowing agent comprises sodium bicarbonate, potassium bicarbonate, ammonium, azodicarbonamide, isocyanate, hydrazine, isopropanol, 5-phenyltetrazole, triazole, 4,4'oxybis(benzenesulfonylhydrazide) (OBSH), trihydrazinetriazine (THT), hydrogen phosphate, tartaric acid, citric acid, and toluenesulfonylsemicarbazide (TSS). In some embodiments, the chemical blowing agent comprises sodium bicarbonate.

[0040] In some embodiments, the blowing agent comprises a physical blowing agent. In some embodiments, the physical blowing agent is a chlorofluorocarbon (CFC), dissolved nitrogen, N 2 , C.H. 4 , H 2 , CO 2 , Ar, pentane, isopentane, hexane, methylene dichloride, and dichlorotetrafluoroethane. In some embodiments, the physical blowing agent comprises dissolved nitrogen.

[0041] According to some embodiments, provided herein is a method for producing a composition comprising a recombinant resilin protein, the method comprising culturing a population of recombinant host cells in a fermentation, the recombinant host cells comprising a vector comprising a secreted resilin coding sequence, the recombinant host cells secreting a recombinant resilin protein encoded by the secreted resilin coding sequence, and purifying the recombinant resilin protein from the fermentation.

[0042] In some embodiments, the recombinant resilin protein is a full-length or truncated native resilin. In some embodiments, the natural resilin is an active ingredient in 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 darlingii, Anopheles spp. In some embodiments, the recombinant resilin protein is from an organism 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 comprises SEQ ID NO:1. In some embodiments, the recombinant resilin protein comprises SEQ ID NO:4.

[0043] In some embodiments, the recombinant resilin protein comprises an alpha mating factor secretion signal. In some embodiments, the recombinant resilin protein comprises a FLAG tag. In some embodiments, the vector comprises multiple secreted resilin coding sequences.

[0044] 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.

[0045] In some embodiments, the recombinant host cells produce recombinant resilin at a rate of greater than 2 mg resilin / g dry cell weight / hour. In some embodiments, the recombinant host cells produce a secreted fraction of recombinant resilin that is greater than 50% compared to the total recombinant resilin protein expressed by the recombinant host cells. In some embodiments, the recombinant host cells secrete recombinant resilin at a rate of greater than 2 mg resilin / g dry cell weight / hour. In some embodiments, greater than 80% of the recombinant resilin is outside the recombinant host cells in the fermentate. In some embodiments, the fermentate comprises at least 2 g recombinant resilin / L.

[0046] In some embodiments, purifying the recombinant resilin protein comprises centrifuging the fermentation to produce a first pellet fraction and a first supernatant fraction, and isolating the recombinant resilin protein from the first pellet fraction, In some embodiments, purifying the recombinant resilin protein further comprises adding a chaotrope to the first pellet fraction to produce a solution in which the recombinant resilin protein is soluble, centrifuging the first pellet fraction containing the chaotrope to produce a second supernatant fraction and a second pellet fraction, and isolating soluble full-length resilin from the second supernatant fraction.

[0047] In some embodiments, provided herein is a vector comprising a secreted resilin coding sequence. 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 an addition, deletion, substitution, or change at an amino acid residue position capable of crosslinking to another resilin.

[0048] In some embodiments, the full length or truncated native resilin is from an organism selected from the group consisting of Drosophila melanogaster, Panama leaf-cutting ant, Acanthoptera spp., Horn fly, Cat flea, Bombus terrestris, Tribolium castaneum, Honey bee, Parasite wasp, Body louse, Anopheles gambiae, Tsetse fly, Ata cephalotes, Anopheles dartingii, Pea aphid, Drosophila niger, Drosophila erecta, Sand fly, Assassin bug, Fire ant, Crex quinquefasciatus, Melon fly, and Trichogramma pretiosum.

[0049] 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 quasi-A repeats. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising one or more B repeats or quasi-B repeats. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising either one or more A repeats or quasi-A repeats, or one or more B repeats or quasi-B repeats, but not both. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising one or more A repeats or quasi-A repeats and one or more B repeats or quasi-B repeats.

[0050] 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 alpha mating factor secretion signal. In some embodiments, the secreted resilin coding sequence comprises a FLAG tag.

[0051] 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 sequences are operably linked to a constitutive or inducible promoter.

[0052] Also provided herein, in some embodiments, is a recombinant host cell comprising one or more vectors comprising a secreted resilin coding sequence. 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, Alcustura adeninivorans, Yarrowia lipolytica, Pichia (Schefersomyces) stipitis, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis.

[0053] In some embodiments, the recombinant host cell comprises three vectors that include a secreted resilin coding sequence.

[0054] In some embodiments, the recombinant host cells produce recombinant resilin at a rate of greater than 2 mg resilin / g dry cell weight / hour. In some embodiments, the recombinant host cells have a secretion fraction of recombinant resilin of greater than 50%. In some embodiments, the recombinant host cells secrete resilin at a rate of greater than 2 mg resilin / g dry cell weight / hour.

[0055] Also provided herein, according to some embodiments, is a fermentation comprising a recombinant host cell comprising one or more vectors that include a secreted resilin coding sequence and a medium suitable for growing the recombinant host cell.

[0056] In some embodiments, the fermentation contains at least 2 g recombinant resilin / L.

[0057] In some embodiments of the fermentation, greater than 80% of the recombinant resilin is outside the recombinant host cell.

[0058] In some embodiments of the fermentation, the recombinant resilin is a full-length recombinant resilin.

[0059] Also provided herein, according to some embodiments, is a composition comprising recombinant resilin derived from a fermentation comprising recombinant host cells comprising one or more vectors comprising a secreted resilin coding sequence and a medium suitable for growing the recombinant host cells, hi some embodiments, the composition comprises at least 60% by weight recombinant resilin.

[0060] In some embodiments, the composition has similar properties compared to a composition that includes a similar amount of natural resilin. In some embodiments, the composition has different properties compared to a composition that includes a similar amount of natural resilin.

[0061] In some embodiments, the composition comprises an elasticity of greater than 50%. 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 elongation at break of greater than 1%. In some embodiments, the composition has an ultimate tensile strength of greater than 0.1 kPa. In some embodiments, the composition has a Shore OO hardness of less than 90. In some embodiments, the composition comprises full-length resilin.

[0062] Also provided herein, according to some embodiments, is a method for producing a composition comprising recombinant resilin, the method comprising culturing a recombinant host cell comprising one or more vectors comprising a secreted resilin coding sequence to produce a fermentate under conditions promoting secretion of the recombinant resilin from the recombinant host cell.

[0063] In some embodiments, the method for producing a composition comprising recombinant resilin further comprises purifying the recombinant resilin to produce full-length native resilin. In some embodiments, purifying the recombinant resilin to produce full-length native resilin comprises centrifuging the fermentation to produce a first pellet fraction and a first supernatant fraction, and isolating the recombinant resilin protein from the first pellet fraction. In some embodiments, isolating the recombinant resilin protein from the first pellet fraction comprises adding a chaotrope to the first pellet fraction to produce a solution in which the recombinant resilin protein is soluble, centrifuging the first pellet fraction containing the chaotrope to produce a second supernatant fraction and a second pellet fraction, and isolating the recombinant resilin protein from the second supernatant fraction.

[0064] In some embodiments, the method for producing a composition comprising recombinant resilin further comprises cross-linking a plurality of said recombinant resilins. In some embodiments, said cross-linking is enzymatic cross-linking. In some embodiments, said cross-linking is photochemical cross-linking. In some embodiments, the recombinant resilin protein comprises a full-length resilin protein.

[0065] Also provided herein, according to some embodiments, is a fermentation comprising a medium and a recombinant host cell, the recombinant host cell comprising a vector, the vector comprising a secreted resilin coding sequence, and the recombinant host cell secretes the recombinant resilin at a rate of at least 2 mg / g dry cell weight / hour. [The present invention 1001] Providing a composition comprising purified recombinant resilin; placing the recombinant resilin in a cross-linking solution containing ammonium persulfate; and incubating said recombinant resilin in said crosslinking solution at a temperature of at least 60° C., thereby producing a crosslinked recombinant resilin solid composition. A method for crosslinking recombinant resilin comprising: [The present invention 1002] 1001. The method of claim 1001, wherein said incubation is carried out for at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, or at least 2 hours. [The present invention 1003] Any of the methods of the present invention, wherein the recombinant resilin in the crosslinking solution is incubated at a temperature of 60°C to 85°C, 70°C to 85°C, or 75°C to 85°C. [The present invention 1004] Any of the methods of the invention, wherein the incubation is carried out for at least 2 hours. [The present invention 1005] Any of the methods of the previous invention, wherein the crosslinking solution does not comprise a photocatalyst or a crosslinking enzyme. [The present invention 1006] Any of the preceding methods of the present invention, wherein the crosslinked recombinant resilin solid composition is stable at room temperature for more than 5 days, more than 10 days, more than 20 days, or more than 40 days. [The present invention 1007] Any of the methods of the invention, wherein the purified recombinant resilin is prepared by recombinantly expressing a gene encoding the recombinant resilin in a modified organism in culture, and purifying the expressed recombinant resilin from the culture. [The present invention 1008] A composition comprising cross-linked recombinant resilin, wherein the recombinant resilin is cross-linked by exposing the resilin to ammonium persulfate and heat. [The present invention 1009] A composition comprising cross-linked recombinant resilin, the composition being free of a cross-linking enzyme. [The present invention 1010] A composition comprising cross-linked recombinant resilin, the composition being free of a photocatalyst. [The present invention 1011] A recombinant resilin composition comprising cross-linked recombinant resilin in a polar non-aqueous solvent. [The present invention 1012] The composition of claim 10, wherein the polar non-aqueous solvent is a protic solvent. [The present invention 1013] The composition of claim 10, wherein the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol. [The present invention 1014] The composition of claim 10, wherein the polar non-aqueous solvent is an aprotic solvent. [The present invention 1015] The composition of claim 1014, wherein the aprotic solvent is DMSO. [The present invention 1016] Any of the compositions according to claims 1011 to 1015, comprising 20 to 40% by weight of resilin. [The present invention 1017] Any of the compositions of claims 1011 to 1016, wherein the resilin is at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a fraction of total resilin as measured by size exclusion chromatography. [The present invention 1018] Any of the compositions of claims 1011 to 1017, wherein the polar non-aqueous solvent is at least 60% by volume, at least 70% by volume, at least 80% by volume, at least 90% by volume, at least 95% by volume, at least 98% by volume, or at least 99% by volume of the solvent. [The present invention 1019] Any of the compositions of claims 1011 to 1018, wherein the recombinant resilin composition has a greater elastic modulus than a similar cross-linked recombinant resilin in an aqueous solvent. [The present invention 1020] Any of claims 1011 to 1019, wherein the recombinant resilin composition comprises a hardness of at least 10 when measured using a Shore 00 durometer according to ASTM D2240. [The present invention 1021] 10. Any of the compositions of claims 1011 to 1020, wherein the recombinant resilin composition comprises a hardness of about 10 to about 50, as measured using a Shore 00 durometer according to ASTM D2240. [The present invention 1022] Any of the compositions of claims 1011 to 1021, wherein the recombinant resilin composition comprises a rebound resilience of about 40% to about 60% as measured by ASTM D7121. [The present invention 1023] Any of the compositions of claims 1011 to 1022, wherein the recombinant resilin composition comprises a compressive stress at 25% of about 6 to about 8 psi as measured by ASTM D575. [The present invention 1024] Any of the compositions of claims 1011 to 1023, wherein the recombinant resilin composition does not undergo an elastic to plastic transition at a compressive force of less than 2 kN when measured in a Zwick compression test. [The present invention 1025] The composition of any one of claims 1011 to 1024, wherein the recombinant resilin composition is a foam material. [The present invention 1026] providing a crosslinked recombinant resilin solid composition in an aqueous solvent; and exchanging the aqueous solvent for a polar non-aqueous solvent. A method for preparing a recombinant resilin solid, comprising: [The present invention 1027] The method of claim 1026, wherein the polar non-aqueous solvent is a protic solvent. [The present invention 1028] The method of claim 1027, wherein the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol. [The present invention 1029] The method of claim 1026, wherein the polar non-aqueous solvent is an aprotic solvent. [The present invention 1030] The method of claim 1029, wherein the aprotic solvent is DMSO. [The present invention 1031] The method of any one of claims 1026 to 1030, wherein the solvent exchange is carried out for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours. [The present invention 1032] 1032. The method of any one of claims 1026 to 1031, wherein the solvent exchange is carried out at about 60°C. [The present invention 1033] Any of the methods of claims 1026 to 1032, wherein the crosslinked recombinant resilin solid composition contains at least 20%, at least 50%, at least 70%, or at least 80% full-length resilin as a fraction of total resilin, as measured by size exclusion chromatography. [The present invention 1034] The method of any one of claims 1026 to 1033, wherein said crosslinked recombinant solid composition is prepared by any one of the methods of claims 1001 to 1007. [The present invention 1035] providing a crosslinked recombinant resilin solid composition comprising an aqueous solvent; and performing a solvent exchange to replace the aqueous solvent with a polar non-aqueous solvent. A method for adjusting the material properties of a solid composition comprising a crosslinked recombinant resilin solid, comprising: [The present invention 1036] The method of claim 1035, wherein replacing the solvent changes the elastic modulus, elasticity, hardness, maximum elastic compressive load, or material lifetime of the crosslinked recombinant resilin solid composition. [The present invention 1037] Providing a composition comprising purified recombinant resilin; placing the recombinant resilin in an aqueous solvent containing ammonium persulfate; incubating said recombinant resilin in said aqueous solvent at a temperature of at least 60° C., thereby producing a crosslinked recombinant resilin solid composition; and exchanging the aqueous solvent for a polar non-aqueous solvent selected from the group consisting of glycerol, propylene glycol, ethylene glycol, and DMSO. A method for preparing a recombinant crosslinked resilin solid, comprising: [The present invention 1038] providing a crosslinked recombinant resilin solid composition in an aqueous solvent; exchanging the aqueous solvent for a polar non-aqueous solvent; and introducing one or more gas bubbles into said crosslinked recombinant resilin solid composition. A method for preparing a recombinant resilin foam comprising: [The present invention 1039] The method of claim 1038, wherein the polar non-aqueous solvent is a protic solvent. [The present invention 1040] The method of claim 1039, wherein the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol. [The present invention 1041] The method of claim 1038, wherein the polar non-aqueous solvent is an aprotic solvent. [The present invention 1042] The method of claim 1041, wherein the aprotic solvent is DMSO. [The present invention 1043] The method of any one of claims 1038 to 1042, wherein the solvent exchange is carried out for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours. [The present invention 1044] The method of any of claims 1038 to 1043, wherein the solvent exchange is carried out at about 60°C. [The present invention 1045] Any of the methods of claims 1038 to 1044, wherein the crosslinked recombinant resilin solid composition contains at least 20%, at least 50%, at least 70%, or at least 80% full-length resilin as a fraction of total resilin, as measured by size exclusion chromatography. [The present invention 1046] The method of any one of claims 1038 to 1045, wherein the crosslinked recombinant solid composition is prepared by the method of any one of claims 1001 to 1007. [The present invention 1047] The method of any one of claims 1038 to 1046, wherein said introducing comprises adding a foaming agent to said crosslinked recombinant resilin solid composition. [The present invention 1048] The method of claim 1047, wherein the blowing agent comprises a chemical blowing agent. [The present invention 1049] The method of the present invention 1048, wherein the chemical blowing agent comprises sodium bicarbonate, potassium bicarbonate, ammonium, azodicarbonamide, isocyanate, hydrazine, isopropanol, 5-phenyltetrazole, triazole, 4,4'oxybis(benzenesulfonylhydrazide) (OBSH), trihydrazine triazine (THT), hydrogen phosphate, tartaric acid, citric acid, and toluenesulfonyl semicarbazide (TSS). [The present invention 1050] The method of claim 1048, wherein the chemical blowing agent comprises sodium bicarbonate. [The present invention 1051] The method of claim 1047, wherein the blowing agent comprises a physical blowing agent. [The present invention 1052] The physical blowing agent is a chlorofluorocarbon (CFC), dissolved nitrogen, N 2 , C.H. 4 , H 2 , CO 2 , Ar, pentane, isopentane, hexane, methylene dichloride, and dichlorotetrafluoroethane. [The present invention 1053] The method of claim 1051, wherein the physical blowing agent comprises dissolved nitrogen. [The present invention 1054] A shoe insole comprising a composition prepared by any one of the methods of the present inventions 1001-1007, 1025-1036, and 1038-1053. [The present invention 1055] A composition comprising a crosslinked resilin composition in a polar non-aqueous solvent, wherein the crosslinked resilin is foamed. [The present invention 1056] The composition of the present invention 1055, wherein said foamed cross-linked resilin comprises at least 50% porosity. [The present invention 1057] The composition of the present invention 1055, wherein said foamed crosslinked resilin has an average cell diameter of from about 0.01 mm to about 3 mm. [The present invention 1058] A shoe insole comprising any one of the compositions of the present invention Nos. 1008 to 1025 and 1055 to 1057. [Brief description of the drawings]

[0066] The above and other objects, features and advantages will become apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

[0067] [Figure 1] 1 shows a schematic diagram of the structure of an exemplary resilin. [Diagram 2] 1 is a flow diagram of a method for producing a composition comprising recombinant resilin. [Diagram 3] 1 is an exemplary map of a vector containing three secreted resilin coding sequences. [Figure 4] Photographs of proteinaceous block copolymers including cross-linked purified recombinant resilin in various shapes and morphologies are shown. [Diagram 5] 1 shows a photograph of compression of a proteinaceous block copolymer containing cross-linked recombinant resilin. [Figure 6] 1 shows total degraded resilin measured by ELISA using an antibody specific for the FLAG tag on resilin for a resilin solid composition crosslinked with ammonium persulfate and heat (AP), a resilin solid composition crosslinked with ammonium persulfate and heat and washed three times to remove excess crosslinking solution (AP 3x wash), and a resilin solid composition enzymatically crosslinked with horseradish peroxidase (HRP). [Figure 7] Photographs of resilin foam containing 10 wt% fumed silica (left) and 5 wt% fumed silica (right) are shown. [Figure 8] A photograph of resilin foam containing 5% by weight fumed silica is shown, with a quarter included for comparison. [Figure 9] Shown are images of glycerol-based and water-based cross-linked resilin compositions after cross-linking (top) and after 7 days (bottom) and one week of storage unsealed. [Figure 10A]1 shows stress strain curves measured on a rheometer for the following crosslinked resilin compositions: 27 wt % resilin in glycerol solvent prepared with a 60% glycerol solvent exchange followed by a 100% glycerol solvent exchange. [Figure 10B] 1 shows stress strain curves measured on a rheometer for the following crosslinked resilin composition: 27 wt % full length resilin in propylene glycol solvent. [Figure 10C] 1 shows stress strain curves measured on a rheometer for the following crosslinked resilin composition: 27% full length resilin in propylene glycol solvent (i.e., approximately 80% full length resilin as a percentage of total resilin). [Figure 11] 1 shows the stress-strain curve of a resilin foam containing 5 wt. % fumed silica. [Figure 12] FIG. 1 shows plots of relative elasticity and modulus measured by a rheometer for each of the following crosslinked resilin solid compositions: 20 wt% resilin, propylene glycol ("A"); 27 wt% resilin, propylene glycol ("B"); 40 wt% resilin, propylene glycol ("C"); resilin coacervate, propylene glycol ("D"); 27 wt% full length resilin, propylene glycol ("E"); 27 wt% resilin, 60-100% glycerol ("F"); 27 wt% resilin, 100% glycerol ("H"); and 27 wt% resilin, ethylene glycol ("I"). [Figure 13] Illustrated are plots of applied force versus strain curves as measured by a Zwick tensile tester for each of the following crosslinked resilin solids: 27 wt% resilin ethylene glycol solid ("A"), 27 wt% resilin propylene glycol solid ("C"), coacervated resilin propylene glycol solid ("D"), 27 wt% resilin 60-100% glycerol solid ("G"), and 27 wt% resilin 100% glycerol solid ("H").

[0068] The figures depict various embodiments of the present disclosure for purposes of illustration only. Those skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be used without departing from the principles described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description The details of various embodiments of the invention are set forth in the description that follows. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

[0070] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0071] As used herein, the terms "a" and "an" and "the" and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0072] The terms "about," "approximately," or "similar" refer to within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which may depend in part on the method by which the value is measured or determined, or the limitations of the measurement system. It should be understood that all ranges and amounts set forth below are approximate and are not intended to limit the invention. When ranges and numerical values ​​are used, they can be approximated to include statistical ranges or measurement errors or variations. In some embodiments, for example, measurements can be plus or minus 10%.

[0073] Amino acids may 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).

[0074] The terms "including," "includes," "having," "has," "with," or variations thereof, are intended to be inclusive in a similar manner as the term "comprising."

[0075] The term "microorganism" as used herein refers to a microorganism and refers to a single-celled organism. As used herein, this 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.

[0076] The term "naturally occurring" as used herein refers to a composition found in nature in its natural, unaltered state.

[0077] The term "optional" or "optionally" means that a feature or structure may or may not be present, or an event or circumstance may or may not occur, and that a description includes instances where the particular feature or structure is present as well as instances where the feature or structure is not present, or instances where an event or circumstance occurs as well as instances where the event or circumstance does not occur.

[0078] As used herein, the term "secreted fraction" refers to the fraction of recombinant resilin that is secreted from a cell compared to the total resilin produced by the cell.

[0079] As used herein, the term "secretion signal" refers to a short peptide that, when fused to a polypeptide, mediates the secretion of that polypeptide from the cell.

[0080] As used herein, the term "secreted resilin coding sequence" refers to a nucleotide sequence encoding a resilin provided herein fused to a secretion signal at the N-terminus and, optionally, fused to a tag peptide or a polypeptide at the C-terminus.

[0081] The term "recombinant" as used herein with respect to a polypeptide (eg, resilin) ​​refers to a polypeptide produced within a recombinant host cell or synthesized from a recombinant nucleic acid.

[0082] As used herein, the term "recombinant host cell" refers to a host cell that contains a recombinant nucleic acid.

[0083] The term "recombinant nucleic acid" as used herein refers to a nucleic acid that is removed from its naturally occurring environment, or that is not associated with all or part of the nucleic acid adjacent or contiguous to it as found in nature, or that is operably linked to a nucleic acid with which it is not linked in nature, or that does not occur in nature, or that contains modifications not found in naturally occurring nucleic acids (e.g., insertions, deletions, or point mutations that are artificially introduced by human intervention, e.g.,) or that is integrated into a chromosome at a heterologous site. The term includes cloned DNA isolates and nucleic acids that contain chemically synthesized nucleotide analogues.

[0084] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include bacteriophages, cosmids, bacterial artificial chromosomes (BAC), and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, in which additional DNA segments can be ligated to the viral genome. Certain vectors can replicate autonomously in the cells into which they are introduced (e.g., vectors with an origin of replication that functions within the cell). Other vectors can integrate into the genome of the cell upon introduction into the cell, thereby replicating together with the cell genome.

[0085] The term "repeat" as used herein with respect to an amino acid or nucleic acid sequence refers to a subsequence (e.g., a sequence that is linked) that is present multiple times within a polynucleotide or polypeptide. A polynucleotide or polypeptide may have a direct repeat of a repeat sequence without any intervening sequence, or may have a non-contiguous repeat of a repeat sequence with an intervening sequence. The term "quasi-repeat" as used herein with respect to an amino acid or nucleic acid sequence is a subsequence that is imprecisely repeated throughout a polynucleotide or polypeptide (i.e., a portion of the quasi-repeat subsequence is variable between quasi-repeats). Repeat polypeptides and DNA molecules (or portions of a polypeptide or DNA molecule) can be composed of either repeat subsequences (i.e., exact repeats) or quasi-repeat subsequences (i.e., inexact repeats).

[0086] The term "natural resilin" as used herein refers to an elastomeric polypeptide or protein produced by an insect. Non-limiting example GenBank accession numbers of natural resilins include the following NCBI SEQ ID NOs: NP995860 (Drosophila melanogaster), NP611157 (Drosophila melanogaster), Q9V7U0 (Drosophila melanogaster), AAS64829, AAF57953 (Drosophila melanogaster), XP001817028 (Trifolium castaneum), and XP001947408 (Pea aphid).

[0087] The term "modified" as used herein refers to a protein or polypeptide sequence that differs in composition from a native protein or polypeptide sequence, while retaining functional properties within 10% of the native protein or polypeptide properties. In some embodiments, the difference between a modified protein or polypeptide and a native protein or polypeptide can be in the primary sequence (e.g., one or more amino acids are deleted, inserted, or substituted) or in post-translational modifications (e.g., glycosylation, phosphorylation). An amino acid deletion refers to the removal of one or more amino acids from a protein. An amino acid insertion refers to one or more amino acid residues that are introduced into a protein or polypeptide. Amino acid insertions may include N-terminal and / or C-terminal fusions, as well as intrasequence insertions of single or multiple amino acids. Amino acid substitutions include non-conservative or conservative substitutions, and conservative amino acid substitution tables are well known in the art (see, for example, Creighton (1984) Proteins. W.H. Freeman and Company (Eds)). In some embodiments, the sequence identity of a variant protein or polypeptide with a naturally occurring protein or polypeptide amino acid or nucleotide 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 acid or nucleotide bases.

[0088] The term "truncated" as used herein refers to a protein or polypeptide sequence that is shorter in length than the native protein or polypeptide. In some embodiments, a truncated protein or polypeptide may be more than 10%, or more than 20%, or more than 30%, or more than 40%, or more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90% of the length of the native protein or polypeptide.

[0089] As used herein, the term "homolog" or "substantial similarity" when referring to a polypeptide, nucleic acid, or fragment thereof, indicates that when optimally aligned with another amino acid or nucleic acid (or its complementary strand), with appropriate amino acid or nucleotide insertions or deletions, there 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% amino acid or nucleotide sequence identity of the amino acid or nucleotide bases as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or Gap, as described above.

[0090] 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, or 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 comprises a native or modified nucleotide sequence encoding (e.g., truncated or concatenated) resilin or a resilin fragment that is heterologously expressed and secreted from a host cell (e.g., isolated from an insect). In a preferred embodiment, the secreted recombinant resilin protein is collected from the extracellular solution of the host cell. In some embodiments, the resilin is in the form of a foam material, e.g., a solid foam.

[0091] As used herein, the term "elastomer" refers to a polymer that has viscoelasticity and usually weak intermolecular forces (except for covalent crosslinks, if any, between the molecules). Viscoelasticity is the property of a material that exhibits both viscous and elastic properties when subjected to deformation, and thus time-dependent strain. Elasticity is associated with the stretching of bonds along the crystal planes of an ordered solid, while viscosity is the result of the diffusion of atoms or molecules within an amorphous material. Thus, viscoelastic elastomers generally have a low Young's modulus and a high strain to failure compared to other materials. Due to the viscous component of the material, viscoelastic materials dissipate energy when a load is applied and then removed. This phenomenon is observed as hysteresis in the stress-strain curve of a viscoelastic material. When a load is applied, there is a specific stress-strain curve, and when the load is removed, the stress-strain curve at unloading is different from the curve at loading. The energy dissipated is the area between the loading and unloading curves.

[0092] As used herein, the term "non-aqueous" refers to a solvent that contains primarily one or more compounds that are not water. This includes compositions that have undergone a solvent exchange process with a solvent in which the percentage of water present as a solvent is reduced overall, i.e., water as a solvent is replaced with non-water molecules. In some embodiments, a non-aqueous solvent is one that contains less than 50% water. A polar non-aqueous solvent, as used herein with respect to the solvent of a crosslinked resilin composition, refers to any non-aqueous solvent in which resilin can be dissolved.

[0093] Recitation of ranges of values ​​herein is merely intended to be used as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually set forth herein.

[0094] Resilin Composition Provided herein are compositions comprising recombinant resilin, and methods for producing them.

[0095] Resilin has many unique properties compared to petroleum-based elastomers. Most notably, resilin has a very high elastic efficiency (i.e., elasticity), meaning that very little of the energy input into deformation is lost as heat. Other desirable properties of resilin include, for example, compressive modulus, tensile modulus, shear modulus, hardness, rebound, and compression set. In addition, resilin is a protein and therefore can be biodegraded, making it more environmentally compatible than petroleum-based polymers. Resilin is also biocompatible and therefore can be used in applications involving human or animal contact. Finally, the mechanical properties of recombinant resilin can be tuned by varying the protein sequence, protein structure, amount of intermolecular crosslinking, and processing variables to generate elastomers designed for specific application areas.

[0096] In some embodiments, provided herein are crosslinked resilin compositions having desirable mechanical properties and methods for producing them. In some embodiments, provided herein are methods for crosslinking resilin compositions that can be efficiently carried out in large batches and form crosslinked resilin solids that are not susceptible to degradation by impurities remaining from the crosslinking reaction. In some embodiments, the crosslinking reaction involves exposure of the resilin to a persulfate, such as ammonium persulfate. Heat can be applied to initiate the crosslinking reaction catalyzed by the persulfate. This type of crosslinking reaction does not leave any photoactive or enzymatic compounds in the composition. Additionally, this crosslinking reaction does not require photoactivation, so large batches can be efficiently produced without the need for light to reach all parts of the crosslinking solution. In some embodiments, the crosslinking occurs in a container or mold such that the resulting recombinant resilin composition has a particular shape or form.

[0097] The crosslinked resilin solid compositions provided herein also include crosslinked resilin compositions that include a polar non-aqueous solvent to provide desired mechanical properties such as modulus, hardness, maximum elastic compressive load, elasticity, and material life / fatigue that are favorable for a particular application. In some embodiments, the compositions are made by performing a solvent exchange with the resilin composition to replace the aqueous solvent with a non-aqueous solvent. Solvents that can be solvent exchanged with the crosslinked resilin include those that dissolve the resilin in its non-crosslinked form.

[0098] In preferred embodiments, the non-aqueous solvent is non-volatile and water-soluble or polar. In some embodiments, the molecular weight of the solvent is about 100 or less. In some embodiments, the polar non-aqueous solvent comprises glycerol, propylene glycol, ethylene glycol, or DMSO. In some embodiments, the solvent exchange is performed using a polar non-aqueous solvent gradient (e.g., 60% to 100% glycerol).

[0099] In some embodiments, the crosslinked resilin compositions described herein can be used to provide compositions with improved physical properties, including, for example, the absorption of energy from an applied force, as desired. In some embodiments, the crosslinked resilin compositions described herein can be used to improve existing products that contain rubber. In particular, some of the crosslinked resilin compositions provided herein can absorb large amounts of force without transitioning to a non-elastic material.

[0100] In some embodiments, the crosslinked resilin compositions provided herein can be used as midsoles. In some embodiments, the crosslinked resilin compositions provided herein can be used as part of the core of a golf ball. In other embodiments, the crosslinked resilin compositions provided herein can be used, for example, in handles or grips for sporting goods such as golf clubs or tennis rackets, as bicycle grips or motorcycle grips, or as grips for tools and industrial applications such as hammers, nail guns, jackhammers, and any other tools where it is desirable to absorb and return energy. In some embodiments, the crosslinked resilin compositions provided herein can be used in bushings or vibration damping, for example, skateboard trucks or hard drive platter vibration dampeners. In some embodiments, the crosslinked resilin compositions can be used as superior materials for wheels of skateboards, rollerblades, or scooters. In some embodiments, the crosslinked resilin compositions provided herein can be used in safety and protective equipment such as padding for protective devices such as helmets, elbow or knee pads, or hard hats, or as a protective outer layer to protect the skin from abrasions.

[0101] In some embodiments, the crosslinked resilin compositions provided herein can be used in automotive parts, for example, suspension components such as bushings or shock absorbers, or interior cushions such as seat bolsters and lumbar supports. In some embodiments, the crosslinked resilin compositions provided herein can be used in tires and inner tubes. In some embodiments, the crosslinked resilin compositions provided herein can be used in super balls. In some embodiments, the crosslinked resilin compositions provided herein can be used in shoe insoles, midsoles, and outsoles. In some embodiments, the crosslinked resilin compositions provided herein can be used in padded mats. In some embodiments, the crosslinked resilin compositions provided herein can be used in some types of gaskets or O-rings. In some embodiments, the crosslinked resilin compositions provided herein can be added to plastic products to increase impact resistance. In some embodiments, the crosslinked resilin compositions provided herein can be used in protective cases such as phone or tablet cases. In some embodiments, the crosslinked resilin compositions provided herein can be used in rubber stamps. In some embodiments, the crosslinked resilin compositions provided herein can be used in rollers. In some embodiments, the crosslinked resilin compositions provided herein can be used in rubber bands.

[0102] In some embodiments, the crosslinked resilin compositions provided herein can be used in the preparation of shoe soles, basement floors, noise protection for music studios, automobile bumpers, cushion pads, door mats, yoga mats, drum pads, window wipers, automobile tires, fire hoses, electrical wiring insulation, rubber bands, rubber ducks, elastic gloves, cookware, rain boots, teethers, bicycle tires, watches, bottles, gaskets, hair ties, flip flops, phone cases, medicine balls, super balls, seals for electronic devices to prevent contamination from water or dust, refrigerator or freezer door seals, seals to prevent air flow in or out of a chamber, trampolines, pacifiers, window seals, Halloween masks, garden hoses, ping pong rackets, conveyor belts, ducts, stamps, balloons, cosmetic compositions for the care and protection of skin and hair, nail compositions, or decorative cosmetics.

[0103] Suitable dermocosmetic compositions are, for example, face tonics, facial masks, such as sheet masks, deodorants, and other cosmetic lotions. Compositions used in decorative cosmetics include, for example, concealer sticks, stage make-up, mascaras and eye shadows, lipsticks, kohl pencils, eyeliners, blushers, powders, and eyebrow pencils.

[0104] Depending on the field of use, the compositions described herein can be applied in a form suitable for skin care, such as, for example, a cream, foam, gel, stick, mousse, milk, spray (pump spray or propellant-containing spray) or lotion.

[0105] FIG. 1 shows an example of a naturally occurring resilin, which contains an N-terminal A domain comprising multiple repeat units with the consensus amino acid sequence YGXP ("A repeat"), where X is any amino acid, a chitin-binding RR-2 (C) domain (Pfam Reference No. 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 comprising multiple repeat units with the consensus amino acid sequence UYZXZ ("B repeat"), where U is glycine or serine, Z is serine, glycine, arginine, or proline, and X is any amino acid. Not all naturally occurring resilins have A, C, and B domains. Natural resilins produced by various insects usually have imprecise repeats (ie, quasi-repeats) in the A and / or B domains, with some amino acid variability between the quasi-repeats.

[0106] In some embodiments, the recombinant resilins provided herein comprise one or more A repeats, hi some embodiments, the recombinant resilins comprise an N-terminal A domain comprising multiple blocks of A repeat and / or quasi-A repeat amino acid subsequences, each having the consensus sequence SXXYGXP (SEQ ID NO:50), where S is serine, X is an amino acid, Y is tyrosine, G is glycine, and P is proline.

[0107] In some embodiments, the recombinant resilins provided herein comprise one or more B repeats, hi some embodiments, the recombinant resilins comprise a C-terminal B domain comprising multiple blocks of B repeat and / or quasi-B repeat amino acid subsequences having the consensus sequences GYZXZZX and / or SYZXZZX, respectively, where G is glycine, Y is tyrosine, Z is serine, glycine, proline, or arginine, S is serine, and X is any amino acid.

[0108] In some embodiments, the recombinant resilins provided herein comprise one or more A repeats, hi some such embodiments, the recombinant resilins comprise between 1 and 100 A repeats, or between 2 and 50 A repeats, or between 5 and 50 A repeats, or between 5 and 20 A repeats.

[0109] In some embodiments, the recombinant resilin has the formula (X 1 -X 2 -X 3 -X 4 ) n (1)(SEQ ID NO:64) and where the brackets indicate repeats or quasi-repeats of the consensus sequence, n denotes the number of A repeats or quasi-A repeats, and is 1 to 100, or 2 to 50, or 5 to 50, or 5 to 20; X 1 is a four amino acid motif, X 1 The first amino acid of is Y and the second amino acid of is X 1 the remaining amino acids are GAP, GLP, GPP, GTP, or GVP, X 2 is a motif of 3 to 20 amino acids in length, X 2 is GGG, GGGG (SEQ ID NO:51), N, NG, NN, NGN, NGNG (SEQ ID NO:52), GQGG (SEQ ID NO:53), GQGN (SEQ ID NO:54), GQGQ (SEQ ID NO:55), GQGQG (SEQ ID NO:56), or contains three or more glycine residues, or 2 More than 50% of the residues in X are either glycine or asparagine, or 2 More than 60% of the residues in X are either glycine or asparagine, or 2 More than 70% of the residues in X are either glycine or asparagine, or 2at least 80% of the residues are either glycine or asparagine, X 3 is a motif of 2-4 amino acids in length, and X 3 is GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG (SEQ ID NO:51); X 4 is a motif of 1-2 amino acids in length, and X 4 is S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS.

[0110] In some such embodiments, the recombinant resilin comprises the motif X 1 , X 2 , X 3 , and X 4 In another embodiment, the recombinant resilin comprises the motif X 1 , X 2 , X 3 , or X 4 , or a combination thereof.

[0111] In some embodiments, the recombinant resilins provided herein comprise one or more B repeats. In some such embodiments, the recombinant resilins comprise between 1 and 100 B repeats, or between 2 and 50 A repeats, or between 5 and 50 A repeats, or between 5 and 20 A repeats.

[0112] In some embodiments, the recombinant resilin has the formula (X 11 -X 12 -X 13 ) m (2)(SEQ ID NO:65) and where the brackets indicate repeats or quasi-repeats of the consensus sequence, m represents the number of B-repeats or quasi-B-repeats and is 1 to 100; X 11is a motif of 1 to 5 amino acids in length, the first amino acid is Y and the remaining amino acids may include GAP, GPP, SSG, or SGG; X 12 is a motif of 2 to 5 amino acids in length, including GQ, GN, RPG, RPGGQ (SEQ ID NO: 57), RPGGN (SEQ ID NO: 58), SSS, SKG, or SN; X 13 is a motif between 4 and 30 amino acids in length and contains GG, DLG, GFG, GGG, RDG, SGG, SSS, GGSF (SEQ ID NO:59), GNGG (SEQ ID NO:60), GGAGG (SEQ ID NO:61), 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.

[0113] In some such embodiments, the recombinant resilin comprises the motif X 11 , X 12 , and X 13 In other such embodiments, the recombinant resilin comprises the motif X 11 , X 12 , or X 13 , or a combination thereof.

[0114] 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, the recombinant resilin comprises one or more A repeats or one or more B repeats, but not both. In some embodiments, the recombinant resilin comprises one or more A repeats, but neither a B repeat nor a C domain. In some embodiments, the recombinant resilin comprises one or more B repeats, but neither an A repeat nor a C domain. In embodiments where the recombinant resilin comprises a C domain, the C domain can be located either N-terminal or C-terminal to the A or B repeats, or between the A and B repeats.

[0115] In some embodiments, the recombinant resilin further comprises the sequence XXEPPVSYLPPS (SEQ ID NO:62), where X is any amino acid. In some such embodiments, the sequence is located N-terminal to the A repeat or B repeat.

[0116] In some embodiments, the recombinant resilin is a full-length native resilin expressed in a non-native environment. In some embodiments, the recombinant resilin comprises a truncated version of a native resilin. In some embodiments, the truncated native resilin comprises at least one A repeat. In some embodiments, the truncated native resilin comprises at least one B repeat. Non-limiting examples of full-length and truncated native resilins are provided as SEQ ID NOs: 1-44. In some embodiments, the recombinant resilin is full-length Seychelles Drosophila melanogaster resilin (SEQ ID NO: 1). In some embodiments, the recombinant resilin is truncated Panama leaf-killer ali resilin (SEQ ID NO: 4). In some embodiments, the recombinant resilin is a full-length or truncated native resilin that is cross-linked in a non-native manner (e.g., less or more cross-links, cross-links via different amino acid residues).

[0117] In some embodiments, the recombinant resilin is a modified full-length or truncated native resilin. In some embodiments, the 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 a full-length or truncated native resilin. In some embodiments, the 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 a full-length Drosophila melanogaster resilin (SEQ ID NO:1). In some embodiments, the 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 a truncated Panama leafkiller ali resilin (SEQ ID NO:4).

[0118] There are several different algorithms known in the art that can be used to measure nucleotide or protein sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, programs of the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA provides alignment and percent sequence identity of the most overlapping regions between the query and search sequences. See, for example, Pearson, Methods Enzymol. 183:63-98, 1990, which is incorporated by reference in its entirety. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with default parameters (word size 6 and NOPAM factor of scoring matrix) or using Gap with default parameters provided in GCG version 6.1, which is incorporated by reference herein. Alternatively, sequences can 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).

[0119] In some embodiments, the modified resilin differs from full-length or truncated native resilin in the amino acid residues that are post-translationally modified (e.g., glycosylated, phosphorylated), such that the modified resilin has one or more post-translational modifications at a different position and / or in a different amount and / or of a different type than full-length or truncated native resilin. In some embodiments, the modified resilin differs from full-length or truncated native resilin in the amino acid residues involved in cross-linking, such that the modified resilin has one or more positions and / or a different amount and / or type of amino acid involved in cross-linking that differ from full-length or truncated native resilin. In some such embodiments, the modified resilin differs from full-length or truncated 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.

[0120] In some embodiments, the recombinant resilin comprises linked native or truncated native resilins or linked modified resilins. In some embodiments, the linked native or truncated native resilins or linked modified resilins comprise at least two A repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the linked truncated native resilins or linked modified resilins comprise at least two B repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

[0121] Protein quantification and purity analysis In some embodiments, the total amount of full-length resilin in the composition is determined by size exclusion chromatography (SEC), reverse-phase high performance liquid chromatography, or other methods known in the art (e.g., quantitative Western blot). In various embodiments, the same or similar assays may be used to assess the relative amounts of monomeric and aggregate forms of full-length resilin.

[0122] As described herein, in certain embodiments, data characterizing the relative amount of the predominant species of resilin (i.e., monomeric full-length resilin) ​​in a resilin composition is determined from size-exclusion chromatography (SEC) as follows: Resilin powder is dissolved in 5 M guanidine thiocyanate and injected onto a Yarra SEC-3000 SEC-HPLC column to separate components based on molecular weight. Refractive index is used as the detection modality. BSA is used as a common protein standard under the assumption that more than 90% of all proteins exhibit dn / dc values ​​(refractive index response factor) within about 7% of each other. Poly(ethylene oxide) is used as a retention time standard, and BSA calibrators are used as check standards to ensure consistent performance of the method. The range corresponding to monomeric full-length resilin is evaluated from 60 to 40 kDa; observed higher peaks that may correspond to aggregated or polymerized full-length resilin are not included in this quantification. The relative percentage of this resilin peak is reported as mass % and area % to determine the amount of full length resilin in the composition as a fraction of the total resilin.

[0123] Crosslinking In some embodiments, the methods provided herein further comprise a step of cross-linking the recombinant resilin to obtain a recombinant resilin composition provided herein (step 1005 in FIG. 2). The recombinant resilin in a desired solvent containing a cross-linking agent can be filled into small molds to control the shape of the resulting solid after cross-linking. An example of the resulting recombinant resilin solid is shown in FIG. 4.

[0124] In some embodiments, cross-linking is accomplished through tyrosine residues. Resilin has a tyrosine residue every 15-24 amino acids throughout most of its length. In some embodiments, cross-linking of resilin produces dityrosine and trityrosine cross-links in the resilin to form a resilin solid.

[0125] In other embodiments, cross-linking is achieved through lysine residues. In some embodiments, cross-linking is achieved through cysteine ​​residues. In some embodiments, cross-linking is achieved using transglutaminase (see, e.g., 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, cross-linking is achieved 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).

[0126] In some embodiments, recombinant resilin is crosslinked via enzymatic crosslinking (e.g., using horseradish peroxidase). While this method can effectively crosslink large solutions of resilin, the resulting crosslinked product contains active enzymes covalently incorporated into the crosslinked resilin solid. This creates a radical chain reaction that degrades the protein backbone of resilin.

[0127] In other embodiments, the recombinant resilin is crosslinked via photochemical crosslinking (see, e.g., 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).

[0128] This crosslinking reaction also results in the incorporation of active catalyst into the resilin solid that is difficult to completely dialyze out of the material, and is not efficient for reactions that use relatively high catalyst loadings and can be difficult to photoactivate through the mold.

[0129] Provided herein is a new recombinant resilin crosslinking chemistry that prevents degradation and creates a solid material that has certain mechanical properties that are favorable for certain applications where the amount and form of energy absorption is important.

[0130] In a preferred embodiment, recombinant resilin is crosslinked via a solvent containing ammonium persulfate and the application of heat (e.g., incubation for about 2.5 hours at a temperature of about 80° C.) This crosslinking reaction is efficient, leaves no active catalyst in the resilin solid, and results in a composition with less protein degradation.

[0131] In some embodiments, the final concentration of ammonium persulfate for the resilin crosslinking reaction is about 30-40 mM. In some embodiments, the crosslinking solution containing ammonium persulfate and resilin is incubated (i.e., exposed to heat) for at least 15 minutes, at least 30 minutes, at least 60 minutes, at least 90 minutes, or at least 2 hours to promote sufficient crosslinking to form a solid with desired mechanical properties. In some embodiments, other persulfate salts are used.

[0132] In some embodiments, the heat applied to the ammonium persulfate and resilin composition solution is at least 40° C., at least 45° C., at least 50° C., at least 55° C., at least 60° C., at least 65° C., at least 70° C., at least 75° C., or at least 80° C. In some embodiments, the heat applied to the ammonium persulfate and resilin crosslinking solution is 50° C. to 90° C., or 60° C. to 90° C., or 70° C. to 90° C., or 75° C. to 85° C., or 70° C. to 80° C. In some embodiments, the crosslinking solution does not include an enzyme catalyst. In some embodiments, the crosslinking solution does not include a photoactive catalyst.

[0133] In some embodiments, the crosslinking solution comprises at least 1%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% resilin by weight of the total crosslinking solution, 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%, 30% to 100%, 90%, 80%, 70%, 60%, 50%, or 40%, 40% to 100%, 90%, 80%, 70%, 60%, or 50%, 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% to 100%. In a preferred embodiment, resilin is dissolved in the crosslinking solution to a concentration of 20% to 30% resilin by weight. In some embodiments, the total resilin contains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% full-length resilin as measured by size exclusion chromatography (SEC).

[0134] Solvent exchange resilin solid Crosslinked resilin can be formed in aqueous solvents, resulting in compositions with low hardness and modulus that are less suitable for certain applications where energy absorption and stiffness are desired. In some embodiments, solvent exchange is performed on the crosslinked resilin composition to replace the aqueous solvent with a polar non-aqueous solvent to provide the desired material properties. In some embodiments, the polar non-aqueous solvent comprises glycerol, propylene glycol, ethylene glycol, or DMSO. In some embodiments, the non-aqueous solvent is water miscible. In some embodiments, the non-aqueous solvent is a non-volatile solvent. In some embodiments, the non-aqueous solvent comprises molecules with multiple alcohol functional groups. In some embodiments, the non-aqueous solvent comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of molecule(s) with a molecular weight less than about 100 g / mol. As described herein, solvent exchange can be used to tailor the material properties of the crosslinked resilin composition, including modulus, hardness, maximum elastic compressive load, elasticity, and material life / fatigue. Solvents that can undergo solvent exchange with the crosslinked resilin include those that dissolve the resilin in its uncrosslinked form.

[0135] In some embodiments, the solvent exchange replacing the aqueous solvent of the resilin with a polar non-aqueous solvent is carried out in the presence of heat, for example at a temperature of about 60° C. In some embodiments, the solvent exchange process is carried out in a solution containing at least 1×, at least 2×, at least 5×, at least 10×, or at least 20× the volume of the exchange solvent relative to the resilin solid. In some embodiments, the solvent exchange is carried out for at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours. In some embodiments, glycerol, propylene glycol, ethylene glycol, or DMSO are used as the exchange solvent for the crosslinked resilin solid composition.

[0136] In some embodiments, the choice of exchange solvent and the concentration used are selected from the solvent design to achieve a desired tunable mechanical property, such as stiffness, which can be selected depending on the desired application (e.g., shoe sole, golf ball, etc.).

[0137] In some embodiments, the crosslinked resilin composition formed in the non-aqueous solvent by the above solvent exchange process is stable at room temperature for at least 10 days, at least 20 days, at least 30 days, at least 40 days, or at least 50 days. In some embodiments, the crosslinked resilin composition formed in the non-aqueous solvent by the above solvent exchange process is resistant to mold growth for at least 10 days, at least 20 days, at least 30 days, at least 40 days, or at least 50 days. In some embodiments, the crosslinked resilin composition formed in the non-aqueous solvent by the above solvent exchange process is resistant to dehydration for at least 7 days or at least 14 days. In contrast, aqueous-based crosslinked resilin solids have been observed to completely dehydrate within 2 weeks and grow mold within 5-14 days when stored in an open environment under ambient conditions. Additionally, a 10 mm x 5 mm disk of aqueous-based resilin solid breaks when subjected to a force of 220 lbs, whereas a 10 mm x 5 mm disk of glycerol-based resilin solid does not break under a force of 220 lbs.

[0138] In some embodiments, the relative material properties between successively tested materials can be tested using a rheometer. For example, a crosslinked resilin composition can be formed in a cylindrical mold, and the resulting resilin cylinder is subjected to a compression test using a rheometer. The recombinant resilin cylinder can be compressed from a starting height of 7.3 mm (average width of 5.4 mm) to less than 0.66 mm without breaking. Upon release of the compression load, the cylinder returns to a height of 6.7 mm (average width of 5.6 mm), as shown in FIG. 5.

[0139] In some embodiments, the polar non-aqueous solvent comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% by volume of the polar non-aqueous composition of the solvent. In some embodiments, the solvent comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% by volume of glycerol, propylene glycol, ethylene glycol, or DMSO.

[0140] In some embodiments, the recombinant resilin compositions provided herein have a greater elastic modulus than cross-linked recombinant resilin in an aqueous solvent.

[0141] In some embodiments, the crosslinked resilin compositions provided herein have a Shore OO hardness of 50 or more, 40 or more, 30 or more, 20 or more, 10 or more, 10 to 50, 40, 30, or 20, 20 to 50, 40, or 30, 30 to 50, or 40, or 40 to 50. In some embodiments, the hardness measurement of the resilin is performed according to ASTM D2240. In some embodiments, the recombinant resilin composition comprises a hardness of at least 10, as measured using a Shore OO durometer according to ASTM D2240. In some embodiments, the recombinant resilin composition comprises a hardness of at least 30, as measured using a Shore OO durometer according to ASTM D2240. In some embodiments, the recombinant resilin composition comprises a hardness of about 10 to about 50, as measured using a Shore OO durometer according to ASTM D2240. In some embodiments, the hardness of the crosslinked resilin composition is comparable to that of an orthopedic sole.

[0142] In some embodiments, the recombinant resilin composition comprises a rebound resilience of about 40% to about 60% as measured by ASTM D7121.

[0143] In some embodiments, the recombinant resilin composition comprises a compressive stress at 25% of about 6 psi to about 8 psi as measured by ASTM D575.

[0144] In some embodiments, the recombinant resilin composition does not undergo an elastic to plastic transition at a compressive force of less than 2 kN as measured by the Zwick compression test.

[0145] In some embodiments, the resilin solid material properties such as elasticity, compressive modulus, tensile modulus, shear modulus, elongation at break, ultimate tensile strength, hardness, stiffness, and rebound can be adjusted based on the solvent used and the manner in which the solvent exchange is performed. For example, glycerol can be exchanged into the resilin solid with or without a gradient, resulting in different material properties. The concentration of resilin in the resilin solid and the amount of full length resilin as a fraction of the total resilin can also be adjusted to affect the material properties of the crosslinked resilin solid composition.

[0146] In some embodiments, as described herein, a solvent exchange replacing an aqueous resilin composition (i.e., a cross-linked resilin composition in an aqueous solvent) with a polar non-aqueous resilin composition (i.e., a cross-linked resilin composition in a polar non-aqueous solvent) results in a stiffer material with similar elasticity and similar stretchability.

[0147] In some embodiments, the compositions provided herein comprise at least 5% by weight, at least 10% by weight, at least 15% by weight, at least 20% by weight, at least 25% by weight, at least 30% by weight, at least 35% by weight, at least 40% by weight, at least 45% by weight, at least 50% by weight, at least 55% by weight, at least 60% by weight, at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight, between 10% and 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight, 30% by weight, or 20% by weight, between 20% by weight and 100% by weight, 90% by weight, %, 80%, 70%, 60%, 50%, 40% or 30% by weight, between 30% and 100%, 90%, 80%, 70%, 60%, 50% or 40% by weight, between 40% and 100%, 90%, 80%, 70%, 60% or 50% by weight, between 50% and 100%, 90%, 80%, 70%, or 60% by weight, between 60% and 100%, 90%, 80% or 70% by weight, between 70% and 100%, 90% or 80% by weight, between 80% and 100% or 90% by weight, or between 90% and 100% by weight. The recombinant resilin can be an identical recombinant resilin or a mixture of recombinant resilins having at least two different amino acid sequences.

[0148] In some embodiments, the compositions provided herein comprise at least 5% by weight, at least 10% by weight, at least 15% by weight, at least 20% by weight, at least 25% by weight, at least 30% by weight, at least 35% by weight, at least 40% by weight, at least 45% by weight, at least 50% by weight, at least 55% by weight, at least 60% by weight, at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight, between 10% and 100%, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight, 30% by weight, or between 20% and 100% by weight. The recombinant resilin composition may comprise between 00%, 90%, 80%, 70%, 60%, 50%, 40% or 30% by weight, between 30% and 100%, 90%, 80%, 70%, 60%, 50% or 40% by weight, between 40% and 100%, 90%, 80%, 70%, 60% or 50% by weight, between 50% and 100%, 90%, 80%, 70%, or 60% by weight, between 60% and 100%, 90%, 80% or 70% by weight, between 70% and 100%, 90% or 80% by weight, between 80% and 100% or 90% by weight, or between 90% and 100% by weight.

[0149] In some embodiments, the recombinant resilin composition is a foamed material. In some embodiments, the crosslinked resilin composition is a foamed material, e.g., a foamed solid. In some embodiments, the foamed material comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% porosity. As described herein, porosity refers to the percentage of the interstitial volume of a material relative to the volume of its mass. In some embodiments, the density of the foamed materials described herein is at least ⅓ of the density of a non-foamed material, e.g., at least ½, at least ¾, or at least twice the density of a non-foamed crosslinked resilin composition. In some embodiments, the average diameter of the bubbles in the foamed materials described herein is from about 0.01 mm to about 3 mm, e.g., from about 0.02 mm to about 1 mm, from about 0.05 mm to about 2 mm, from about 0.1 mm to about 3 mm, from about 0.2 mm to about 4 mm, from about 0.5 mm to about 5 mm, from about 1 mm to about 1.5 mm, or from about 1.5 mm to about 2 mm.

[0150] In some embodiments, the recombinant resilin composition comprises one or more layers of foam material. In some embodiments, the one or more layers of foam material are arranged in a desired order, e.g., in a sequential or alternating configuration, where a support layer is placed between two layers of foam material. The one or more layers of foam material may be arranged by any method known in the art, e.g., spray drying, injection molding. In some embodiments, the one or more layers of foam material comprise one or more voids. The topography of each layer and the tendency of the one or more layers of foam material to overlap together may be different. In some embodiments, the voids may provide channels on one or more sides to help direct air flow and provide additional absorption capacity.

[0151] Mechanical properties In other embodiments, the compositions provided herein have different properties compared to compositions comprising crosslinked resilin. In some embodiments, the compositions provided herein have similar properties compared to synthetic elastic materials. Non-limiting examples of such properties include elasticity, compressive modulus, tensile modulus, shear modulus, hardness, rebound, and compression set. Parameters that can be modified to obtain compositions with specific mechanical properties include, for example, the length and / or sequence of the recombinant resilin, the degree and / or type of post-translational modification of the recombinant resilin, the degree and / or type of crosslinking of the recombinant resilin, and the nature of the solvent of the crosslinked resilin composition.

[0152] In some embodiments, many different types of tension and compression systems can be used to perform stress-strain measurements on elastomer samples to measure mechanical properties such as ultimate tensile strength, compression modulus, tensile modulus, shear modulus, elongation at break, and elasticity. The resulting stress-strain curves, including those with hysteresis, can be measured in tension or compression. In some embodiments, tension and compression testing systems can apply strain to the sample and measure the resulting force using a load cell. In some embodiments, mechanical properties can be measured on a macroscopic scale (e.g., using a macroscopic compression tester), microscopic, or nanoscale (e.g., using atomic force microscopy (AFM) or nanoindentation measurements). In some embodiments, compressive mechanical properties of elastomers can be measured according to standard ASTM D575-91 (2012) Standard Test Method for Rubber Properties in Compression. Mechanical measurements of elastomers in tension can be performed using ASTM D412-15a Standard Test Method for Vulcanized Rubber and Thermoplastic Elastomers - Tension. In some embodiments, the tear strength of elastomers can 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, bonded, and molded elastomers can be performed using ASTM D3574-11 Standard Test Method for Flexible Cellular Materials-Slab, Bonded, and Molded Urethane Foams. In some embodiments, the mechanical properties of elastomers can be measured using ASTM D5992-96(2011) Standard Guide for Dynamic Testing of Vulcanized Rubber and Rubber-Like Materials Using Vibration Methods.

[0153] In some embodiments, the compositions provided herein have an elasticity of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater 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%.

[0154] 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 can be measured as defined by ASTM D575-91 (2012) Standard Test Method for Rubber Properties in Compression.

[0155] 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, 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.

[0156] In some embodiments, the 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.

[0157] In some embodiments, the compositions provided herein have an elongation at break of greater than 1%, greater than 10%, greater than 50%, greater than 100%, greater than 300%, or greater 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%.

[0158] In some embodiments, the compositions provided herein have an ultimate 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.

[0159] In some embodiments, mechanical properties such as hardness and compressive modulus can be measured using indentation and nanoindentation measurement systems. In some embodiments, indentation measurements utilizing a tip that penetrates into a sample to a given amount of strain are used to measure the hardness and compressive modulus of resilin, and the resulting force is measured using a load cell. In some embodiments, a variety of tip geometries can be used, including Vickers and Berkovich shaped tips. In some embodiments, hardness measured by the indentation technique is characterized by the relationship Hardness=(Peak Force) / (Contact Area).

[0160] In some embodiments, the hardness of polymers, elastomers, and rubbers can be measured using a hardness tester. In some embodiments, the hardness of elastomers can be measured using standard ASTM D2240, which recognizes 12 different durometer scales that use combinations of specific spring forces and indenter configurations. The most common scales are the Shore OO, A, and D hardness scales. The hardness scale ranges from 0 to 100, with 0 being a softer material and 100 being a harder material.

[0161] In some embodiments, the compositions provided herein have a Shore OO 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 to 90. In some embodiments, the hardness measurement of resilin is performed according to ASTM D2240.

[0162] As used herein, the term "rebound" refers to a measure of specific elasticity. In some embodiments, rebound can be measured using a number of different tools, including a pendulum tool and a drop ball. In a pendulum type measurement, the RB, commonly referred to as the rebound ratio, is determined from the following formula: TIFF2024129078000002.tif11128

[0163] The rebound resilience can be calculated as follows: TIFF2024129078000003.tif10128, where h = height of peak of rebound, H = initial height. Rebound resilience can also be determined by measuring the angle of rebound. Some examples of test methods for determining the rebound of elastomers are ASTM D2632-15 and ASTM D7121-05(2012).

[0164] In some embodiments, the compositions provided herein have a rebound of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater 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, resilin rebound measurements are performed according to ASTM D2632-15, or ASTM D7121-05(2012).

[0165] As used herein, the term "compression set" refers to a measure of the permanent deformation remaining after the applied force is removed. In some embodiments, compression set can be measured in a variety of ways, including compression set under a constant force in air (referred to as compression set A), compression set under a constant deflection in air (referred to as compression set B), and compression set under a constant deflection in air that takes into account the hardness of the material (referred to as compression set C). Compression set A (C A ) is expressed by the following formula, CA =[(t o -t i ) / t o ] × 100 (wherein, t o is the original specimen thickness, and t i is the thickness of the specimen under test). Compression set B (C B ) is C B =[(t o -t i ) / (t o -t n )] × 100 (where t o is the original specimen thickness, and t i is the thickness of the specimen after testing, and t n (where θ is the thickness of the spacer or specimen under test.) Some examples of test methods for determining the compression set of elastomers are ASTM D3574-11 and ASTM D395-16.

[0166] In some embodiments, the compositions provided herein have a compression set A or compression set B of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater 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 measurements for resilin are performed according to ASTM D3574-11 and ASTM D395-16.

[0167] The processing and shaping of resilin into products can take many forms depending on the various applications. Thus, the compositions provided herein can have any shape and form, including but not limited to, gels, porous sponges, films, machinable solids, cast forms, molded forms, and composite materials.

[0168] 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 can be determined using a pycnometer using alcohol instead of water to remove air bubbles. In some embodiments, the density of an elastomer can be determined using a hydrostatic method. As used herein, the term "compressed volume density" refers to the ratio of the sample mass to the compressed volume of the sample, where "compressed volume" is defined as the final equilibrium volume achieved with an elastomer sample when subjected to a compressive force sufficient to move freely until it completely conforms to the surrounding shape of a piston-cylinder test chamber enclosure. In some embodiments, the compressed volume density of an elastomer can be determined using a compression volume density meter.

[0169] In some embodiments, the compositions provided herein are administered in a dose of 0.5 mg / cm 3 ~2.0mg / cm 3 , or 1.0 mg / cm 3 ~1.5mg / cm 3 , or 1.1 mg / cm 3 ~1.4mg / cm 3 , or 1.2 mg / cm 3 ~1.35mg / cm 3 In some embodiments, the determination of the density or compressed volume density of the elastomer can be performed using ASTM D297-15 Standard Test Methods for Rubber Products - Chemical Analysis.

[0170] The compositions provided herein have several applications, including but not limited to, applications in aerospace, automobiles, sporting goods, anti-vibration, footwear, and clothing, among others. Some applications in these categories are listed as non-limiting examples. Due to the desirable elastic efficiency, resilin can be used as an energy storage device (e.g., rubber bands) to store and recover mechanical energy. Automotive suspension systems can be improved by applying resilin bushings to maintain more contact of the tire on the road when going over bumps and through potholes at high speeds. In addition, there are several sporting goods applications of resilin with variously tailored mechanical properties, including golf ball cores, tennis racket grips, golf club grips, and table tennis paddles.

[0171] An especially interesting application is footwear due to the unique properties of the resilin compositions provided herein. As an insole or midsole, resilin can improve the comfort and bioefficiency of shoes by cushioning the foot strike and restoring much of the energy from the foot strike as forward momentum. As a midsole, resilin can be the entire midsole or encapsulated within another material to complement the properties (e.g., abrasion or wear resistant materials, or materials tailored for traction). Resilin midsoles can also contain multiple resilin materials with differently tailored mechanical properties that work together to provide enhanced performance (e.g., a softer heel strike area and firmer arch support).

[0172] Vectors, host cells, and fermentations Further provided herein are vectors encoding recombinant resilin, recombinant host cells comprising such vectors, and fermentations comprising such recombinant host cells and recombinant resilin.

[0173] In some embodiments, the vectors provided herein contain a secreted resilin coding sequence that encodes a resilin polypeptide fused at the N-terminus to a secretion signal and, optionally, at the C-terminus to a tag peptide or polypeptide, hi some embodiments, the vectors contain a secreted resilin coding sequence that is codon-optimized for expression in a particular host cell.

[0174] Suitable secretion signals are those that mediate the secretion of polypeptides in recombinant host cells provided herein. Non-limiting examples of suitable secretion signals are the secretion signals of alpha mating factor (α-MF) from Saccharomyces cerevisiae, acid phosphatase (PHO1) from Pichia pastoris, and phytohemagglutinin (PHA-E) from Phaseolus vulgaris. Further secretion signals are known in the art or can be identified by identification of proteins secreted by the host cell, followed by genomic analysis of the secreted proteins and identification of the untranslated N-terminal sequences (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).

[0175] The resilin encoded by the secreted resilin coding sequence can be further fused to a tag peptide or polypeptide. Non-limiting examples of tag peptides or polypeptides include affinity tags (i.e., peptides or polypeptides that bind to a particular agent or matrix), solubilization tags (i.e., peptides or polypeptides that aid in proper folding of the protein and prevent precipitation), chromatography tags (i.e., peptides or polypeptides that alter the chromatographic properties of the protein to obtain different resolution for a particular separation technique), epitope tags (i.e., peptides or polypeptides that are bound by antibodies), fluorescent tags (i.e., peptides or polypeptides that emit higher wavelengths of light when excited with shorter wavelengths of light), chromogenic tags (i.e., peptides or polypeptides that absorb in a particular segment of the visible light spectrum), enzyme substrate tags (i.e., peptides or polypeptides that are substrates for a particular enzymatic reaction), chemical substrate tags (i.e., peptides or polypeptides that are substrates for a particular chemical modification), or combinations thereof. Non-limiting examples of suitable affinity tags include maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag, SBP tag, strep tag, and calmodulin tag. Non-limiting examples of suitable solubility tags include thioredoxin (TRX), poly(NANP), MBP, and GST. Non-limiting examples of chromatography tags include polyanionic amino acids (e.g., the FLAG tag). TIFF2024129078000004.tif4128) and polyglutamic acid 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 derivatives thereof. Non-limiting examples of chromogenic tags include non-fluorescent members of the GFP-like family of proteins (e.g., BlitzenBlue, DonnerMagenta; DNA2.0, Newark, Calif.). Non-limiting examples of enzyme substrate tags include peptides or polypeptides that contain a lysine in the sequence suitable for biotinylation (e.g., AviTag, biotin carboxy carrier protein [BCCP]). Non-limiting examples of chemical substrate tags include substrates suitable for reaction with FIASH-EDT2. C-terminal peptide or polypeptide fusions to resilin can be cleavable (e.g., by TEV protease, thrombin, factor Xa, or enteropeptidase) or non-cleavable.

[0176] In some embodiments, the vector comprises a single secreted resilin coding sequence. In other embodiments, the vector comprises two or more (e.g., three, four, or five) secreted resilin coding sequences. In some such embodiments, the secreted resilin coding sequences are identical. In other such embodiments, at least two of the secreted resilin coding sequences are not identical. In embodiments in which at least two of the secreted resilin coding sequences are not identical, the at least two secreted resilin coding sequences may differ from each other in the resilin and / or secretion signal and / or any tag peptides or polypeptides that encode them.

[0177] In some embodiments, the vector comprises a promoter operably linked to the secreted resilin coding sequence to drive expression of the secreted resilin coding sequence. The promoter can be a constitutive promoter or an inducible promoter. In some embodiments, induction of the inducible promoter occurs via glucose repression, galactose induction, sucrose induction, phosphate repression, thiamine repression, or methanol induction. Suitable promoters include promoters that mediate expression of proteins in the recombinant host cells provided herein. Non-limiting examples of suitable promoters include AOX1 promoter, GAP promoter, LAC4-PBI promoter, T7 promoter, TAC promoter, GCW14 promoter, GAL1 promoter, lambda PL promoter, lambda PR promoter, beta-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 drive expression of the secreted resilin coding sequence are known in the art.

[0178] In some embodiments, the vector comprises a terminator operably linked to the secreted resilin coding sequence to effect termination of transcription of the secreted resilin coding sequence. Suitable terminators include terminators that terminate transcription in the 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 effect termination of transcription of the secreted resilin coding sequence are known in the art.

[0179] In embodiments in which the vector comprises more than one resilin coding sequence, the two or more resilin coding sequences may be operably linked to the same promoter and / or terminator, or to two or more different promoters and / or terminators.

[0180] The vectors provided herein may further include elements suitable for propagation of the vector in a recombinant host cell. Non-limiting examples of such elements include bacterial origins of replication and selection markers (e.g., antibiotic resistance genes, auxotrophic markers). Bacterial origins of replication and selection markers are known in the art. In some embodiments, the selection marker is a drug resistance marker. Drug resistance markers allow cells to detoxify exogenously added drugs that would otherwise kill the cells. Examples of drug resistance markers include, but are not limited to, resistance to antibiotics such as ampicillin, tetracycline, kanamycin, bleomycin, streptomycin, hygromycin, neomycin, and Zeocin™. In some embodiments, the selection marker is an auxotrophic marker. Auxotrophic markers allow cells to synthesize an essential component (usually an amino acid) while growing in a medium lacking the essential component. A selectable auxotrophic gene sequence includes, for example, hisD, which allows growth in histidine-free medium in the presence of histidinol. Other suitable selectable markers for the vectors of the present invention include the bleomycin resistance gene, metallothionein gene, hygromycin B-phosphotransferase gene, AURI gene, adenosine deaminase gene, aminoglycoside phosphotransferase gene, dihydrofolate reductase gene, thymidine kinase gene, and xanthine-guanine phosphoribosyltransferase gene.

[0181] The vector of the present invention may further comprise a targeting sequence that directs the incorporation of the secreted resilin coding sequence into a specific location within the genome of the host cell. Non-limiting examples of such targeting sequences include nucleotide sequences that are identical to nucleotide sequences present in the genome of the host cell. In some embodiments, the targeting sequence is identical to a repetitive element in the genome of the host cell. In some embodiments, the targeting sequence is identical to a transposable element in the genome of the host cell.

[0182] In some embodiments, provided herein is a recombinant host cell comprising a vector as described herein. In some embodiments, the vector is stably integrated into the genome (e.g., chromosome) of the recombinant host cell, for example, by homologous recombination or targeted integration. Non-limiting examples of suitable sites for genomic integration include the Ty1 locus of the Saccharomyces cerevisiae genome, the rDNA and HSP82 loci of the Pichia pastoris genome, and transposable elements whose copies are interspersed throughout the genome of the recombinant host cell. In other embodiments, the vector is not stably integrated into the genome of the recombinant host cell, but rather is extrachromosomal.

[0183] Recombinant host cells can be of mammalian, plant, algal, fungal, or microbial origin. Non-limiting examples of suitable fungi include methylotrophic yeasts, filamentous yeasts, Alcustura adeninivorans, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Candida versatilis, Debaryomyces hansenii, Candida niger var. awamori ... hansenii, Endothia parasitica, Eremothecium ashbyii, Fusarium moniliforme, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor miehei var. Cooney et Emersonii,Cooney et Emerson, Mucor pusillus Lindt, Penicillium roquefortii, Pichia methanolica, Pichia pastoris (Komagataella phaffii), Pichia (Schefersomyces) stipitis, Rhizopus niveus, Rhodotorula sp., Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces budding yeast, Saccharomyces chevalieri, Saccharomyces diastaticus diastaticus, Saccharomyces ellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum, Sporidiobolus johnsonii, Sporidiobolus salmonicol salmonicolor, Sporobolomyces roseus, Trichoderma reesi, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, Zygosaccharomyces rouxii, and their derivatives and hybrids.

[0184] 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, Lactobacillus acidophilus, and the like. acidophilus, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactococcus lactis, Lactococcus lactis Lancefield group N, Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroides strain NRRL B-512(F), Micrococcus lysodeikticus, Spirulina, Streptococcus cremoris, Streptococcus lactis lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilusthermophilus, Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes, Streptomyces rubiginosus, Xanthomonas campestris, and derivatives and hybrids thereof. 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" is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "recombinant host cell" as used herein.

[0185] In some embodiments, the recombinant host cell comprises genetic modifications that improve production of the 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.

[0186] The recombinant host cell provided herein is produced by transforming a suitable cell of origin with the vector provided herein.In such a transformation, the vector can be circular or linear.The transformation of the recombinant host cell containing the vector can be easily identified, for example, by expressing the drug resistance or auxotrophy marker encoded by the vector, which allows for the selection of or for the growth of the cell, or by other means (for example, by detecting the luminescent peptide contained in the vector, by molecular analysis of individual recombinant host cell colonies, for example, by restriction enzyme mapping, PCR amplification, or sequence analysis of the isolated extrachromosomal vector or chromosomal integration site).

[0187] In some embodiments, the recombinant host cells provided herein can produce high titers of the recombinant resilin provided herein. In some such embodiments, the recombinant host cells can produce greater than 2 mg resilin / g dry cell weight / hr, greater than 4 mg resilin / g dry cell weight / hr, greater than 6 mg resilin / g dry cell weight / hr, greater than 8 mg resilin / g dry cell weight / hr, greater than 10 mg resilin / g dry cell weight / hr, greater than 12 mg resilin / g dry cell weight / hr, greater than 14 mg resilin / g dry cell weight / hr, greater than 16 mg resilin / g dry cell weight / hr, greater than 18 mg resilin / g dry cell weight / hr, greater than 20 mg resilin / g dry cell weight / hr. The recombinant resilin is produced at a rate of greater than 25 mg resilin / g dry cell weight / hour, or greater than 30 mg resilin / g dry cell weight / hour, 2 to 40, 30, 20, 10, or 5 mg resilin / g dry cell weight / hour, 5 to 40, 30, 20, or 10 mg resilin / g dry cell weight / hour, 10 to 40, 30, or 20 mg resilin / g dry cell weight / hour, 20 to 40 or 30 mg resilin / g dry cell weight / hour, or 30 to 40 mg resilin / g dry cell weight / hour. In other such embodiments, the recombinant host cell is capable of producing greater than 2 mg resilin / g dry cell weight / hr, greater than 4 mg resilin / g dry cell weight / hr, greater than 6 mg resilin / g dry cell weight / hr, greater than 8 mg resilin / g dry cell weight / hr, greater than 10 mg resilin / g dry cell weight / hr, greater than 12 mg resilin / g dry cell weight / hr, greater than 14 mg resilin / g dry cell weight / hr, greater than 16 mg resilin / g dry cell weight / hr, greater than 18 mg resilin / g dry cell weight / hr, or greater than 20 mg resilin / g dry cell weight / hr. , greater than 25 mg resilin / g dry cell weight / hour, or greater than 30 mg resilin / g dry cell weight / hour, 2 to 40, 30, 20, 10, or 5 mg resilin / g dry cell weight / hour, 5 to 40, 30, 20, or 10 mg resilin / g dry cell weight / hour, 10 to 40, 30, or 20 mg resilin / g dry cell weight / hour, 20 to 40 or 30 mg resilin / g dry cell weight / hour, or 30 to 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.

[0188] In some embodiments, the recombinant host cells provided herein have a high secretion fraction of the recombinant resilin provided herein, in some such embodiments, the recombinant host cells secrete a fraction of the recombinant resilin of 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%.

[0189] The production and secretion of recombinant resilin can be affected by the number of copies of the secreted resilin coding sequence contained in the recombinant host cell and / or the rate of transcription of the secreted resilin coding sequence contained in the recombinant host cell. In some embodiments, the recombinant host cell comprises a single secreted resilin coding sequence. In other embodiments, the recombinant host cell comprises two or more (e.g., three, four, five, or more) secreted resilin coding sequences. In some embodiments, the recombinant host cell comprises a secreted resilin coding sequence operably linked to a strong promoter. A non-limiting example of a strong promoter includes the pGCW14 promoter of Pichia pastoris. In some embodiments, the recombinant host cell comprises a secreted resilin coding sequence operably linked to a medium promoter. A non-limiting example of such a medium promoter includes the pGAP promoter of Pichia pastoris. In some embodiments, the recombinant host cell comprises a coding sequence encoding resilin under the control of a weak promoter.

[0190] The fermentations provided herein comprise a recombinant host cell as described herein and a medium suitable for growing the recombinant host cell.

[0191] The fermentation product is obtained by culturing the recombinant host cells in a medium that provides nutrients required by the recombinant host cells for cell survival and / or growth, and secretion of recombinant resilin. Such a medium usually contains an excess of a carbon source. 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, ribose, 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.

[0192] In some embodiments, the fermentate comprises at least 1%, at least 5%, at least 10%, at least 20%, or at least 30%, 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% by weight of the total fermentate. The recombinant resilin may be present in an amount of 0%, 30% to 100%, 90%, 80%, 70%, 60%, 50% or 40%, 40% to 100%, 90%, 80%, 70%, 60% or 50%, 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% or 90% to 100% by weight.

[0193] In some embodiments, the fermentate is at least 2 g / L, at least 5 g / L, at least 10 g / L, at least 15 g / L, at least 20 g / L, at least 25 g / L, or at least 30 g / L, 2 g / L to 300 g / L, 200 g / L, 100 g / L, 90 g / L, 80 g / L, 70 g / L, 60 g / L, 50 g / L, 40 g / L, 30 g / L, 20 g / L, or 10 g / L, 10 ... 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 to 300g / L, 200g / L, 100g / L, 90g / L, 80g / L, 70g / L, 60g / L, 50g / L, 40g / L, or 30g / L, 3 ... / L, 70g / L, 60g / 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, 80 or 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.

[0194] Methods for Producing Recombinant Resilin Further provided herein are methods for the production of the recombinant resilins described herein.

[0195] Methods are generally carried out according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, e.g., 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 Press, 1999.

[0196] In some embodiments, novel methods are utilized to secrete resilin extracellularly from a host cell. In some embodiments, the methods include constructing a vector containing a secreted resilin coding sequence (step 1001 in FIG. 2), transforming the vector into a host cell (step 1002 in FIG. 2), and then culturing the recombinant host cell, which secretes resilin extracellularly (step 1003 in FIG. 2). In some embodiments, the method comprises: greater than 2 mg resilin / g dry cell weight / hour, greater than 4 mg resilin / g dry cell weight / hour, greater than 6 mg resilin / g dry cell weight / hour, greater than 8 mg resilin / g dry cell weight / hour, greater than 10 mg resilin / g dry cell weight / hour, greater than 12 mg resilin / g dry cell weight / hour, greater than 14 mg resilin / g dry cell weight / hour, greater than 16 mg resilin / g dry cell weight / hour, greater than 18 mg resilin / g dry cell weight / hour, greater than 20 mg resilin / g dry cell weight / hour, greater than 25 ... The method includes secreting resilin out of the cell at a rate of greater than 30 mg resilin / g dry cell weight / hour, 2 to 40, 30, 20, 10, or 5 mg resilin / g dry cell weight / hour, 5 to 40, 30, 20, or 10 mg resilin / g dry cell weight / hour, 10 to 40, 30, or 20 mg resilin / g dry cell weight / hour, 20 to 40 or 30 mg resilin / g dry cell weight / hour, or 30 to 40 mg resilin / g dry cell weight / hour. In some embodiments, the secreted resilin is then purified (step 1004 of FIG. 2 ), and the purified resilin is crosslinked to form an elastomer (step 1005 of FIG. 2 ). In some embodiments, the method provided herein includes transforming a cell with a vector provided herein to obtain a recombinant host cell provided herein (step 1002 of FIG. 2 ). Methods for transforming cells with 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 transfection. Those skilled in the art can select one or more suitable methods for transforming cells with the vectors provided herein based on their knowledge in the art that certain techniques for introducing vectors work better for certain types of cells.

[0197] In some embodiments, the method further comprises culturing a recombinant host cell provided herein in a medium under suitable conditions to obtain a fermentation product provided herein (step 1003 in FIG. 2). In some embodiments, the conditions and medium are suitable to promote secretion of recombinant protein from the recombinant host cell into the medium. Suitable media for use in these methods are known in the art, as are suitable culture conditions. Exemplary details of culturing 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.

[0198] In some embodiments, the method further comprises purifying the secreted recombinant resilin from the fermentation provided herein to obtain the recombinant resilin provided herein (step 1004 in FIG. 2). Purification can occur by a variety of methods known in the art for purifying secreted proteins from fermentation. A common step of such a method includes centrifugation (to remove cells), followed by precipitation of the protein using a precipitant or other suitable kosmotrope (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. Additionally, 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 subjected to coacervate formation.

[0199] In various embodiments, methods for purifying secreted recombinant proteins from fermentations can include various centrifugation steps combined with solubilization of the protein in whole cell broth or cell pellets with known chaotropes such as urea or guanidine thiocyanate.

[0200] In some embodiments, the recombinant resilin protein can be purified by centrifuging the whole cell broth to produce a first pellet of cells and a first supernatant, and extracting the first supernatant to produce a clarified cell broth. The first supernatant is then precipitated using ammonium sulfate and centrifuged to produce a second pellet and a second supernatant (discarded). The second pellet is then resuspended in PBS for dialysis. In some embodiments, the dialyzed solution is then subjected to high temperature to denature proteins that are less stable than the target recombinant protein. The denatured proteins are then removed by centrifuging the dialyzed and denatured solution to produce a third pellet and a third supernatant. The third supernatant is retained from the denatured solution, and then cooled to induce coacervate formation, inducing phase separation into a dense lower layer and an upper layer containing the target recombinant resilin protein. Samples purified by this method can be referred to as "CCB" samples. In some embodiments, multiple coacervate formation is performed by retaining the lower layer and incubating the lower layer at a lower temperature to induce further phase separation.

[0201] In some embodiments, recombinant resilin protein can be purified by centrifuging the whole cell broth to produce a first pellet of cells and proteins proximate to the cells (e.g., attached to the cells, on the surface of the cells) and / or insoluble proteins (e.g., protein aggregates) and a first supernatant, then discarding the first supernatant to obtain the first pellet. The first pellet can then be resuspended in guanidine thiocyanate to solubilize the recombinant resilin protein. The resuspension can then be centrifuged again to produce a second pellet and a second supernatant. The second supernatant can then be dialyzed against PBS, subjected to high temperature to denature proteins other than recombinant resilin, and centrifuged to produce a third pellet and a third supernatant. In some embodiments, the third supernatant can then be subjected to coacervate formation by cooling, resulting in phase separation into a dense lower layer and an upper layer containing recombinant resilin. Samples purified by this method can be referred to as "gel layer" samples. In some embodiments, multiple coacervate formation is performed by retaining the lower layer and incubating the lower layer at a lower temperature to induce further phase separation.

[0202] In some embodiments, recombinant resilin protein can be purified by centrifuging the whole cell broth to produce a pellet and a supernatant, then discarding the supernatant to obtain a pellet of cells and proteins proximate to the cells (e.g., attached to the cells, on the surface of the cells) and / or insoluble proteins (e.g., protein aggregates). The pellet of cells is then resuspended in guanidine thiocyanate to solubilize proteins proximate to the cells. The resuspension is centrifuged again to produce a second pellet of cells and a second supernatant. The second supernatant is then precipitated with ammonium sulfate and centrifuged to produce a third pellet and a third supernatant. The third pellet is then suspended in guanidine thiocyanate, then dialyzed against PBS, subjected to high temperature to denature proteins other than recombinant resilin, and centrifuged to produce a fourth supernatant and a fourth pellet. The fourth supernatant is then subjected to coacervate formation by cooling to cause phase separation. Samples purified by this method can be referred to as "precipitated gel layer" samples.

[0203] In some embodiments, recombinant resilin protein can be purified by adding urea to whole cell broth to solubilize the protein, then centrifuging the whole cell broth to produce a first pellet and a first supernatant. The first supernatant is then precipitated using ammonium sulfate and centrifuged to produce a second pellet and a second supernatant. The second supernatant is discarded, and the second pellet is then resuspended in guanidine thiocyanate, dialyzed against PBS, then subjected to high temperature to denature proteins other than recombinant resilin, and centrifuged again to produce a third pellet and a third supernatant. The third supernatant is then cooled to coacervate the third supernatant, inducing phase separation into a dense lower layer and an upper layer containing recombinant resilin. Samples purified by this method can be referred to as "urea WCBE" samples.

[0204] In some embodiments, recombinant resilin protein can be purified by centrifuging the whole cell broth to produce a first pellet and a first supernatant, then discarding the first supernatant to obtain a first pellet of cells and proteins proximate to the cells (e.g., attached to the cells, on the surface of the cells) and / or insoluble proteins (e.g., protein aggregates). The first pellet of cells is then resuspended in guanidine thiocyanate to solubilize the proteins. The resuspension is centrifuged again to produce a second pellet of cells and a second supernatant. The second supernatant is dialyzed against PBS and then centrifuged to produce a heavy phase of proteins, a light phase of supernatant, and a membrane that separates the heavy phase from the light phase. The heavy phase of proteins is then separated by discarding the light phase and membrane. Samples purified by this method can be referred to as "dense layer" samples.

[0205] In some embodiments, the recombinant resilin composition is a foamed material. In some embodiments, the method of preparing a recombinant resilin foam includes providing a cross-linked recombinant resilin solid composition in an aqueous solvent, exchanging the aqueous solvent with a polar non-aqueous solvent, and introducing one or more gas bubbles into the cross-linked recombinant resilin solid composition. Any method of introducing gas bubbles known in the art may be used herein. For example, methods of introducing gas bubbles include, but are not limited to, vortexing, mixing, adding yeast, and chemical reaction. In some embodiments, the introduction of one or more gas bubbles may occur simultaneously with the cross-linked recombinant resilin solid composition being provided. In some embodiments, the introduction of one or more gas bubbles may occur after the cross-linked recombinant resilin solid composition is provided.

[0206] A blowing agent is typically introduced into a polymeric material to create a polymer foam. According to one embodiment, a chemical blowing agent is mixed with the polymer. The chemical blowing agent undergoes a chemical reaction in the polymeric material, usually under conditions where the polymer melts, causing the formation of a gas. Chemical blowing agents are generally low molecular weight organic compounds that decompose at a certain temperature and release a gas such as nitrogen, carbon dioxide, or carbon monoxide.

[0207] Exemplary chemical blowing agents include, but are not limited to, sodium bicarbonate, potassium bicarbonate, ammonium, azodicarbonamide, isocyanate, hydrazine, isopropanol, 5-phenyltetrazole, triazole, 4,4'oxybis(benzenesulfonylhydrazide) (OBSH), trihydrazinetriazine (THT), hydrogen phosphate, tartaric acid, citric acid, and toluenesulfonylsemicarbazide (TSS).

[0208] In some embodiments, foaming agents, thickening agents, and / or hardening agents are added to the recombinant resilin solid. Exemplary foaming agents include, but are not limited to, xanthan gum, sodium dodecyl sulfate, ammonium lauryl sulfate, bovine serum albumin. Exemplary thickening agents include, but are not limited to, fumed silica and xanthan gum. Exemplary hardening agents include, but are not limited to, aliphatic polyamines, fatty polyamides, aromatic polyamine hardeners, anhydride hardeners, boron trifluoride hardeners, and hardener (dicyandiamide).

[0209] According to another embodiment, a physical blowing agent, i.e., a fluid that is a gas under ambient conditions, is injected into a molten polymer stream to form a mixture. The mixture is subjected to a pressure reduction, causing the blowing agent to expand and form bubbles (cells) within the polymer. In some embodiments, the pressure required is from about 500 psi to about 2000 psi, e.g., from about 600 psi to about 1000 psi, from about 700 psi to about 1500 psi, and from about 800 psi to about 2000 psi. In some embodiments, the pressure required is about 500 psi.

[0210] Exemplary physical blowing agents include chlorofluorocarbons (CFCs), dissolved nitrogen, N 2 , C.H. 4 , H 2 , CO 2 , Ar, pentane, isopentane, hexane, methylene dichloride, and dichlorotetrafluoroethane.

[0211] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the invention is not intended to be limited to the above Description, but is as set forth in the appended claims.

[0212] In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or otherwise clear from the context. A claim or description containing "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0213] It is also noted that the term "comprising" is intended to be open, allowing, but not requiring, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0214] When ranges are given, the endpoints are included. Moreover, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to contemplate any specific value or subrange of the described ranges of different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

[0215] All cited sources, for example, references, publications, databases, database entries, and techniques cited herein, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between the statements of a cited source and this application, the statements of this application shall control.

[0216] The section and table headings are not intended to be limiting. EXAMPLES

[0217] Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but, of course, some experimental error and deviation should be allowed for.

[0218] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0219] Example 1: Production of recombinant resilin Pichia pastoris recombinant host cells secreting recombinant resilin were generated by transforming a HIS+ derivative of GS115 (NRRL Y15851) Pichia pastoris ( Komagataella faphii ) with a vector containing the secreted resilin coding sequence.

[0220] The vectors each contained three resilin coding sequences fused in frame to an N-terminal secretion signal (alpha mating factor leader and pro sequence) and, in some cases, a C-terminal 3xFLAG tag (SEQ ID NO:45) (see FIG. 3). Each of the secreted resilin coding sequences was flanked by a promoter (pGCW14) and a terminator (tAOX1 pA signal). The vectors further contained targeting regions that could direct integration of the three secreted resilin coding sequences into the HSP82 locus of the Pichia pastoris genome, dominant resistance markers for selection of bacterial and yeast transformants, and a bacterial origin of replication.

[0221] Resilin coding sequences were obtained from searches of the scientific literature and public sequence databases. The nucleotide sequences were translated into amino acid sequences and then codon optimized. Both full-length and truncated resilin sequences were selected. The selected secreted resilin coding sequences are shown in Table 1.

[0222] Table 1. Exemplary full-length and truncated resilin amino acid sequences and recombinant host strains TIFF2024129078000005.tif192154

[0223] The vectors were transformed into Pichia pastoris using electroporation to generate a host strain containing three integrated copies of each secreted resilin coding sequence. Transformants were plated onto YPD agar plates supplemented with antibiotics and incubated at 30 °C for 48 h.

[0224] Clones 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 agitation at 1,000 rpm. Samples were removed, recombinant host cells were pelleted by centrifugation, and the supernatants were collected and run on SDS-PAGE gels for analysis of resilin content via Coomassie gels (for polypeptides containing 3xFLAG tags) and Western blot analysis. For FLAG-tagged proteins, the remaining cultures were used to inoculate duplicate minimal medium cultures for ELISA measurements. One replicate was pelleted and the supernatant was measured directly. The second replicate was extracted with guanidine thiocyanate and both intracellular and extracellular fractions were measured.

[0225] Recombinant resilin from multiple species was successfully expressed in Pichia pastoris recombinant host cells, which secreted up to 90% of the recombinant resilin produced.

[0226] SEQ ID NO:49 of the sequence listing provides the full-length Drosophila Seychelles resilin sequence (Ds_ACB) that is expressed with a signal sequence that is subsequently cleaved. The first sequence (italics) is the alpha mating factor precursor protein signal sequence (SEQ ID NO:46) that is cleaved twice after transcription by signal peptidase followed by cleavage by Kex2. The second sequence (bold) is the EAEA repeat (SEQ ID NO:47) that is cleaved by Ste13 (SEQ ID NO:47). The third sequence (lowercase) corresponds to the full-length Drosophila Seychelles resilin (SEQ ID NO:1). The fourth sequence (bold and italics) corresponds to the linker sequence (SEQ ID NO:46). The fifth sequence (underlined) corresponds to the 3X FLAG tag (SEQ ID NO:45).

[0227] Edman sequencing confirmed that the N-terminus of the protein sequence of the approximately 110 kDa band corresponded to the full-length Drosophila melanogaster resilin sequence. Specifically, N-terminal sequencing showed that the N-terminus corresponded to "EAEA" (SEQ ID NO:47) or "GRPE" (SEQ ID NO:63), the full-length Drosophila melanogaster resilin sequence with or without the EAEA repeats (SEQ ID NO:47), respectively.

[0228] Example 2: Purification of recombinant resilin Non-FLAG-tagged Ds_ACB and Ae_A recombinant resilin polypeptides were purified as follows: strains RMs1221 (expressing Ds_ACB) and RMs1224 (expressing Ae_A) were grown in 500 mL of BMGY in flasks at 30° C. for 48 hours with agitation at 300 rpm.

[0229] The purification protocol of Lyons et al. (2007) was adapted. 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 volume of phosphate buffered saline (PBS) and the resuspended sample was dialyzed against PBS to remove salt. The dialyzed sample was then heated to denature the native proteins and the denatured proteins were removed by centrifugation. The retained supernatant contained the purified resilin polypeptide. Optionally, the retained supernatant was allowed to cool, which resulted in coacervate formation, resulting in the concentration of the lower phase and dilution of the upper phase.

[0230] A second set of samples was prepared by centrifuging the whole cell broth to produce a first pellet of cells and proteins proximate to the cells (e.g., attached to the cells, on the surface of the cells) and / or insoluble proteins (e.g., protein aggregates) and a first supernatant, then discarding the first supernatant to obtain the first pellet. The first pellet was resuspended in guanidine thiocyanate to solubilize Ds_ACB. The resuspension was centrifuged again to produce a second pellet and a second supernatant. The second supernatant was then dialyzed against PBS, 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 coacervate formation by cooling to produce phase separation into a dense lower layer and an upper layer containing Ds_ACB. This is also referred to herein as the gel layer separation, or coacervate forming sample. In some gel layer samples, multiple coacervate formations were performed by retaining the lower layer and incubating it at a lower temperature to induce further phase separation.

[0231] Table 2. Stability of cross-linked resilin TIFF2024129078000006.tif75128

[0232] Example 3: Purification, secretion, and cross-linking of recombinant resilin Purified resilin was crosslinked via one of three methods: photocrosslinking (adapted from Elvin et al. 2005), enzymatic crosslinking (adapted from Qin et al. 2009), or as described herein (i.e., ammonium persulfate and heat).

[0233] photocrosslinking For photocrosslinking, resilin protein was mixed with ammonium persulfate and tris(bipyridine)ruthenium(II) ([Ru(bpy)3]2+). After exposing the mixture to bright white light, the mixture formed a rubbery solid.

[0234] Specifically, a 10 mM Ru(II) photocatalyst (CAS 50525-27-4) stock solution and a 100 mM ammonium persulfate stock solution were prepared. Purified resilin powder was dissolved in phosphate buffered saline to a resilin concentration of 20-30 wt% to prepare a resilin stock solution.

[0235] 2 μL of 10 mM Ru(II) photocatalyst solution and 2 μL of 100 mM ammonium persulfate were added to 10 μL of resilin stock solution and mixed to form a photocrosslinking solution. Air bubbles were removed from the photocrosslinking solution by centrifugation, and the photocrosslinking solution was poured into a mold.

[0236] The photocrosslinking solution was then exposed to white light (white LED floodlight, Zochlon USPT100W 110V) for 30 seconds to 1 minute.

[0237] enzymatic crosslinking For enzymatic cross-linking, resilin protein was mixed with horseradish peroxidase (HRP) and hydrogen peroxide, and after the mixture was incubated at 37° C., the mixture formed a rubbery solid.

[0238] Specifically, a 10 mg / mL stock of horseradish peroxidase (type 1, P8125 Sigma) in water (HRP stock) and 100 mM H 2 O 2 Resilin stock solution was prepared by dissolving purified resilin powder in phosphate buffered saline to a resilin concentration of 20-30 wt%.

[0239] 6 μL of HRP stock was added to 21 μL of resilin stock solution and mixed. 2 O 2 3 μL of stock was added and mixed by vortexing to form the enzymatic crosslinking solution. Air bubbles were removed from the enzymatic crosslinking solution by centrifugation, and the enzymatic crosslinking solution was poured into a mold while maintaining the solution at 4° C. To effect crosslinking, the enzymatic crosslinking solution was incubated at 37° C. for 15 minutes. The resulting crosslinked resilin composition contains active enzyme covalently bound to the resilin solid.

[0240] Persulfates and thermal crosslinking The purified resilin was cross-linked using ammonium persulfate and heat.

[0241] Specifically, a resilin stock solution was prepared by dissolving purified resilin powder in phosphate buffered saline to a resilin concentration of 20-30 wt%. 4 μL of 200 mM ammonium persulfate in water was mixed with 18 μL of the resilin stock solution to a final concentration of 36 mM ammonium persulfate. Air bubbles were removed from the persulfate crosslinking solution by centrifugation, and the persulfate crosslinking solution was poured into a mold.

[0242] To effect crosslinking, the persulfate crosslinking solution was incubated in a closed moist environment for 2.5 hours at 80° C. The resulting crosslinked resilin composition results in a composition with no active catalyst remaining in the resilin solids and less protein degradation.

[0243] Degradation of HRP Crosslinking Composition The stability of the crosslinked samples was evaluated over time by determining the period during which each crosslinked sample remained solid through daily observations. Table 3 shows the time as a solid for each crosslinked sample prepared by photocrosslinking (Ru+hv), enzymatic crosslinking (HRP), and crosslinking with ammonium persulfate and heat (AP+heat). As shown in Table 3, at room temperature, the resilin compositions crosslinked with photocrosslinking or ammonium persulfate had a much longer period of stability than the resilin compositions crosslinked by enzymatic crosslinking.

[0244] Table 3. Degradation of resilin compositions crosslinked by various crosslinking procedures TIFF2024129078000007.tif66156

[0245] Total degraded resilin for ammonium persulfate cross-linked resilin compositions (AP) and enzymatically cross-linked resilin compositions (HRP) measured by ELISA is shown in Figure 6. Increased degradation and secretion of resilin fragments from the cross-linked resilin compositions is observed in the HRP samples compared to the AP samples.

[0246] This data indicates that ammonium persulfate cross-linking is preferred over enzymatic cross-linking for preparing stable resilin compositions. In enzymatically cross-linked resilin compositions, the cross-linking enzyme remains in the composition, resulting in degradation of the composition.

[0247] Example 4: Stability of cross-linked full-length resilin and resilin mixtures Resilin samples produced with various levels of degradation products and full-length resilin were subjected to enzymatic cross-linking as described above. The stability of the cross-linked samples was evaluated over time by determining the period during which each cross-linked sample remained solid through daily observations. Table 4 shows the time as a solid for each cross-linked sample. As shown in Table 4, samples containing full-length resilin were stable for a longer period than samples without full-length resilin.

[0248] Table 4. Stability of cross-linked resilin TIFF2024129078000008.tif75128

[0249] Example 5: Resilin Foam Crosslinking resilin in the presence of a thickening additive, e.g., fumed silica, and sodium bicarbonate, resulted in foamed resilin with a relatively uniform cell distribution. Varying the amount of fumed silica affects the average cell size, providing a means to tailor the foamed resilin solids. The results are shown in Table 5.

[0250] Table 5. Resilin foam data TIFF2024129078000009.tif60128

[0251] A solution of resilin was prepared. 0.81 g of resilin was dissolved in 2.19 mL of PBS, pH 7.4. A foaming agent was added and the solution was vortexed to introduce air bubbles, which were then crosslinked by heat or light. 50 mg of xanthan gum was added to the solution, followed by 0.65 mL of 225 mM ammonium persulfate. Xanthan gum was used as a foaming agent, as it also increased the viscosity, which helped to remove air bubbles in the solution. The solution was then vortexed and heated at 80°C for 3.5 hours.

[0252] In some samples, resilin was crosslinked using ruthenium(II) and white light. In one such experiment, 0.405 g of resilin lot 3 was dissolved in 1.10 mL of PBS. Then, 0.3 mL of 100 mM ammonium persulfate and 0.3 mL of 10 mM Ru(II) catalyst were added. In other samples, 0.405 g of resilin lot 3 was dissolved in 1.10 mL of PBS. Then, 25 mg of xanthan gum was added, followed by 0.3 mL of 100 mM ammonium persulfate and 0.3 mL of 10 mM Ru(II) catalyst.

[0253] In some samples, sodium bicarbonate was used as a chemical foaming agent. Sodium bicarbonate was added to the resilin solution to induce crosslinking. In some samples, fumed silica, a thickening agent, was added. Too much sodium bicarbonate (more than 33 mg / mL) inhibited gelation. Using 6-20 mg / mL of sodium bicarbonate in the resilin solution resulted in a resilin solid with large bubbles after 3.5 hours at 80 °C. Addition of 4-10 wt% fumed silica, a thickening agent, resulted in a resilin foam with uniformly distributed bubbles (Figures 7 and 8). Too little fumed silica produced a semi-foamed resilin solid, and adding more than 5 wt% fumed silica resulted in a foamed resilin with smaller bubbles (Figure 7). The bubble diameters of resilin foams with 5 wt% fumed silica were 0.2-2 mm. The cell diameter of the resilin foam containing 10 wt % fumed silica was 0.05-0.2 mm.

[0254] Specifically, 0.27 g of resilin lot 3 was dissolved in 0.73 mL of PBS at pH 7.4. 37, 18, 10, or 6 mg of sodium bicarbonate was then added to the solution, followed by 0.22 mL of 225 mM ammonium persulfate and 0-10 wt% fumed silica. The solutions were vortexed and heated at 80 °C for 3.5 h. Experiments were performed in capped 2 mL tubes.

[0255] For some samples, an ISCO pump was used to introduce dissolved nitrogen at either 1600 psi or 500 psi while crosslinking resilin at 83° C. Crosslinking at these pressures proceeded at a slower rate than crosslinking at atmospheric pressure, presumably due to the increased amount of dissolved oxygen.

[0256] Specifically, 1.1 mL of 225 or 550 mM ammonium persulfate in water was added to 5 mL of 27 wt% resilin in PBS. This solution was then centrifuged at 7197 rcf for 5 minutes and added to the ISCO pump. The pump was then connected to house nitrogen and the ISCO pump was set to a maximum capacity of 266 mL, purged with nitrogen for 3 minutes, and then sealed. The pump was then set to either 500 psi or 1600 psi and heated at 83°C for 2-6 hours. After releasing the pressure, the resilin was heated at 83°C for an additional 1-2 hours.

[0257] Example 6: Solvent exchange and comparison of cross-linked resilin in aqueous and polar non-aqueous solvents As described in detail below, cross-linked mixtures of recombinant resilin in aqueous solvents were subjected to solvent exchange to replace the solvent with a polar non-aqueous solvent to form compositions with material properties more suitable for particular applications.

[0258] A resilin powder was prepared containing 19% by weight full-length resilin as determined by SEC (size exclusion chromatography), with the remaining powder containing partially degraded resilin.

[0259] The resilin powder was then dissolved in 1× phosphate buffered saline (PBS) and the reported weight percentage of resilin is weight / weight (w / w). 1 g of a 27 wt% solution of resilin was prepared by adding 0.27 g of resilin to 0.73 g of the PBS solution. The resilin solution was then cross-linked in a mold to form a water-based resilin solid composition. The resilin solid formed was a disk 15 mm in diameter by 5 mm in height.

[0260] To generate the glycerol-based resilin solids, the water-based solids are exchanged into 20x the volume of 60% glycerol (60:40 glycerol:water (v:v)) in a sealed container for 16 hours at 60° C. The solids are then exchanged into 20x the volume of glycerol in an unsealed container for 16 hours at 60° C.

[0261] Samples of the water-based and glycerol-based resilin solids were stored unsealed at room temperature for one week. As shown in Figure 9, the water-based resilin solids dehydrated after only one week, while the glycerol-based resilin solids maintained their original shape without any visible decomposition or dehydration.

[0262] Example 7: Solvent exchange and comparison of the properties of several polar non-aqueous resilin solvents Resilin solids in polar non-aqueous solvents were prepared from cross-linked resilin in 1× phosphate buffered saline (PBS) as follows.

[0263] Resilin solids in propylene glycol solvent were prepared by exchanging the resilin solids in 20× volume of propylene glycol in an unsealed container at 60° C. for 16 hours or 2 days (i.e., 2-day incubation).

[0264] Resilin solids in 60-100% glycerol solvent were prepared by exchanging the resilin solids in 20x volume of 60% glycerol (60:40 glycerol:water (v:v)) in a sealed container for 16 h at 60 °C. The solids were then exchanged into 20x volume of glycerol in an unsealed container for 16 h at 60 °C.

[0265] Resilin solids in 100% glycerol solvent were prepared by exchanging the resilin solids in 20× volume of glycerol in an unsealed container at 60° C. for 16 hours or 2 days (i.e., 2-day incubation).

[0266] Resilin solid in ethylene glycol solvent was prepared by exchanging the resilin solid in 20× volume of ethylene glycol at 60° C. for 16 hours in an unsealed container.

[0267] Resilin solid in propylene glycol solvent was prepared by exchanging resilin solid in 20× volume of propylene glycol at 60° C. for 16 hours in an unsealed container.

[0268] To form resilin solids based on purification by coacervate formation, a 27% by weight (w / w) resilin solution was incubated at -5°C, resulting in liquid-liquid phase separation. The lower coacervate layer was separated, and ammonium persulfate (APS) powder was mixed into the coacervate to a final concentration of 36 mM APS. This solution was then crosslinked at 80°C for 2.5 hours. The solid was then exchanged following a typical solvent exchange procedure.

[0269] Unless otherwise noted, the resilin powder used was 19% full length by weight as determined by size exclusion chromatography (SEC), with the remainder of the powder consisting of partially degraded resilin. A 27% by weight "full length resilin" sample was prepared from resilin powder that was 87% full length monomeric resilin by weight as determined by SEC.

[0270] To characterize the relative amount of predominant species of resilin in the resilin composition using SEC, resilin powder was dissolved in 5 M guanidine thiocyanate and injected onto a Yarra SEC-3000 SEC-HPLC column for separation into components based on molecular weight. Refractive index was used as the detection modality. BSA was used as a common protein standard under the assumption that >90% of all proteins exhibit dn / dc values ​​(refractive index response factor) within about 7% of each other. Poly(ethylene oxide) was used as a retention time standard and BSA calibrators were used as check standards to ensure consistent performance of the method. The range corresponding to 60-40 kDa resilin was evaluated and any higher peaks observed that may correspond to aggregated or polymerized resilin were not included in this quantification. To determine the amount of full-length resilin in the composition as a fraction of the total resilin, the relative percentage of this resilin peak was reported as mass% and area%.

[0271] A Malvern Kinexus Rheometer RNX2110 was then used to generate stress-strain curves for each of the compositions made as above. The following protocol was applied at a temperature of 22° C.: Step 1: Initial gap - 6.5 mm, Step 2: Drop to final gap. Steps 1 and 2 were repeated 5 times. The values ​​of the elastic modulus measured with the rheometer are only relative and cannot be compared to materials tested under different conditions or with different equipment.

[0272] The engineering stress was plotted against the engineering strain to generate a stress-strain curve. The engineering stress was calculated from the following equation: Engineering stress = (normal force on the resilin solid in contact with the rheometer geometry (N) / A (mm 2 ))×10 6 The engineering strain is the absolute value of the compressive strain = ABS((current compressed height of the resilin solid (mm) - height of the uncompressed solid (mm)) / height of the uncompressed solid (mm)) * 100.

[0273] Figure 10A shows the stress-strain curves of 27 wt% cross-linked resilin in 60-100% glycerol. Figure 10B shows the stress-strain curves of 27 wt% cross-linked resilin in propylene glycol. Figure 10C shows the stress-strain curves of 27 wt% full-length cross-linked resilin in propylene glycol. For comparison, rheometry data for Dr. Sholl's inserts was also generated.

[0274] As shown in Figure 11, the stress-strain curve of the foamed resilin had a hockey stick shape, which is the result of air pockets in the foamed resilin, which first compress and then compress the resilin material.

[0275] From the rheometry data, the relative modulus and elasticity were determined as shown in Table 5. The relative elasticity (%) was calculated by taking the area under the load curve, dividing it by the area under the unload curve in the engineering stress versus strain plot, and multiplying this value by 100. The relative modulus was calculated by taking the slope of the load curve from 10 to 20% absolute compressive strain.

[0276] As shown, switching the cross-linked resilin solvent from an aqueous to a polar non-aqueous solvent resulted in a stiffer material with similar elasticity and similar stretchability. All cross-linked resilin compositions were elastic under the conditions tested (less than 20 N force).

[0277] The rheometer was also used to measure the elasticity of 5 wt. % foamed resilin produced as described in Example 5, containing fumed silica as the blowing agent in 100% glycerol solvent, and the results are also shown in Table 6.

[0278] Table 6. Relative values ​​of elastic modulus and elasticity of cross-linked resilin in various solvents TIFF2024129078000010.tif79158

[0279] The relative values ​​of elasticity and modulus from Table 6 were plotted as shown in Figure 12. This plot shows the relationship between elasticity and modulus when the crosslinked resilin properties were adjusted by solvent exchange.

[0280] The rheometer was also used to measure the relative elasticity and modulus of separately prepared crosslinked resilin solids in propylene glycol, and the results are shown in Table 7.

[0281] Table 7. Rheometer data for similarly prepared propylene glycol solids. TIFF2024129078000011.tif28170

[0282] A rheometer was also used to measure the relative elasticity and modulus of crosslinked resilin solids in various solvents by applying repeated forces to determine the effect of repeated forces on these parameters, and the results are shown in Table 8.

[0283] Table 8. Rheometer data: Multiple compression elasticity and modulus data TIFF2024129078000012.tif108164

[0284] Example 8: Stiffness of Resilin Solids and Representative Midsoles Crosslinked silk solid compositions were prepared in water, 60-100% glycerol, 100% glycerol, propylene glycol (16 hour incubation), and propylene glycol (2 day incubation as described in Example 6). The stiffness of these materials was tested using a Shore OO durometer (AD-100-00) per ASTM:D-2240. For comparison, Dr. Scholl's insole and a representative midsole were also tested. The results are shown in Table 9.

[0285] Table 9. Stiffness of various resilin compositions vs. representative midsole stiffness TIFF2024129078000013.tif76128

[0286] Example 9: Elastic to Plastic Transition Measured by Zwick Compression Curves Zwick compression data was obtained for 27 wt% resilin ethylene glycol solids ("A"), 27 wt% resilin propylene glycol solids ("C"), coacervate resilin propylene glycol solids ("D"), 27 wt% resilin 60-100% glycerol solids ("G"), and 27 wt% resilin 100% glycerol solids ("H"). Each of these samples was prepared as described in Example 5.

[0287] Specifically, the compression data of the above samples was tested using a Zwick tensile tester equipped with a 5kN load cell and a 5kN compression platen (Zwick / Roell Z5.0). The solid dimensions of each solid were 15mm x 5mm disks. The materials were tested using the D695 Compression Properties of Rigid Plastics compression program at a temperature of 22C (ambient temperature) and 65% humidity (ambient).

[0288] The force versus strain curves measured on a Zwick tensile tester for each solid are shown in Figure 13. No elastic to plastic transition is observed for any of the resilin solids at applied compressive forces up to 2 kN. Instead, if the resilin solids failed under compressive stress, it was due to tensile rather than elastic deformation.

[0289] Example 10: Elasticity and Compression Analysis of Propylene Glycol Resilin Solids The resilience and compressive stress at 25% strain were measured using ASTM D7121 and ASTM D575 tests for 27% by weight propylene glycol exchanged resilin solids prepared as in Example 5. The dimensions of the solids tested were 1.125 inch diameter disks with a height of 0.8 inch. Three different samples of these dimensions were tested. The results are shown in Table 10.

[0290] Table 10: Elasticity and compression data for propylene glycol resilin solids TIFF2024129078000014.tif21160

[0291] The data shown in Table 10 can be compared to other rubbers or elastomers tested with the same ASTM test. The ASTM test numbers are sufficient to reproduce the results. This data and the durometer data are the only data that can be used to directly compare an elastomer to other rubbers.

[0292] Further considerations The above description of embodiments of the present disclosure has been presented for purposes of illustration and is not intended to be exhaustive or to limit the scope of the claims to the precise forms disclosed. Those skilled in the art will appreciate that numerous modifications and variations are possible in light of the above disclosure.

[0293] The language used herein has been selected primarily for readability and instruction, and may not be selected to limit or restrict the subject matter of the present invention. Therefore, the scope of the disclosure is intended to be limited not by this detailed description, but rather by any claims issued in an application based on this specification. Thus, the disclosure of the embodiments is intended to illustrate, but not limit, the scope of the invention, which is set forth in the claims.

[0294] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

[0295] Sequence Listing TIFF2024129078000015.tif212156TIFF2024129078000016.tif230156TIFF2024129078000017.tif190156TIFF2024129078000018.tif205156TIFF2024129078000019.tif193156TIFF2024129078000020.tif211156TIFF2024129078000021.tif177156TIFF2024129078000022.tif159156TIFF2024129078000023.tif237156TIFF2024129078000024.tif193156TIFF2024129078000025.tif211156TIFF2024129078000026.tif238156TIFF2024129078000027.tif229156

[0296] Array information SEQUENCE LISTING <110> BOLT THREADS, INC. <120> CROSS-LINKED ELASTOMERIC PROTEINS IN POLAR NONAQUEOUS SOLVENTS AND USES THEREOF <150> US 62 / 700,197 <151> 2018-07-18 <160> 65 <170> PatentIn version 3.5 <210> 1 <211> 606 <212> PRT <213> Drosophila sechellia <400> 1 Arg Pro Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Ser Asp Ser Tyr 1 5 10 15 Gly Ala Pro Gly Gln Ser Gly Ala Gly Gly Arg Pro Ser Asp Thr Tyr 20 25 30 Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp Ser Tyr Gly 35 40 45 Ala Pro Gly Gln Gly Gln Gly Gln Gly Gln Gly Gln Gly Gly Tyr Gly 50 55 60 Gly Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn 65 70 75 80 Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly 85 90 95 Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly 100 105 110 Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Gly Asn Gly Asn 115 120 125 Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly 130 135 140 Asn Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly 145 150 155 160 Gly Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly 165 170 175 Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn 180 185 190 Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn 195 200 205 Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly 210 215 220 Asn Gly Ser Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ala Gln 225 230 235 240 Gly Gln Gly Gly Phe Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro 245 250 255 Gly Gln Asn Gln Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly 260 265 270 Asn Gly Ser Ala Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser 275 280 285 Gly Pro Gly Gly Arg Pro Ser Asp Ser Tyr Gly Pro Pro Ala Ser Gly 290 295 300 Ser Gly Ala Gly Gly Ala Gly Gly Ser Gly Pro Gly Gly Ala Asp Tyr 305 310 315 320 Asp Asn Asp Glu Pro Ala Lys Tyr Glu Phe Asn Tyr Gln Val Glu Asp 325 330 335 Ala Pro Ser Gly Leu Ser Phe Gly His Ser Glu Met Arg Asp Gly Asp 340 345 350 Phe Thr Thr Gly Gln Tyr Asn Val Leu Leu Pro Asp Gly Arg Lys Gln 355 360 365 Ile Val Glu Tyr Glu Ala Asp Gln Gln Gly Tyr Arg Pro Gln Ile Arg 370 375 380 Tyr Glu Gly Asp Ala Asn Asp Gly Ser Gly Pro Ser Gly Pro Ser Gly 385 390 395 400 Pro Gly Gly Pro Gly Gly Gln Asn Leu Gly Ala Asp Gly Tyr Ser Ser 405 410 415 Gly Arg Pro Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly Tyr Ser Ser 420 425 430 Gly Arg Pro Gly Gly Gln Asp Leu Gly Pro Ser Gly Tyr Ser Gly Gly 435 440 445 Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Gly Tyr Ser Asn Val Lys 450 455 460 Pro Gly Gly Gln Asp Leu Gly Pro Gly Gly Tyr Ser Gly Gly Arg Pro 465 470 475 480 Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Gly Gly Arg Pro Gly 485 490 495 Gly Gln Asp Leu Gly Ala Gly Ala Tyr Ser Asn Gly Arg Pro Gly Gly 500 505 510 Asn Gly Asn Gly Gly Ser Asp Gly Gly Arg Val Ile Ile Gly Gly Arg 515 520 525 Val Ile Gly Gly Gln Asp Gly Gly Asp Gln Gly Tyr Ser Gly Gly Arg 530 535 540 Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Ser Gly Arg Pro 545 550 555 560 Gly Gly Arg Pro Gly Gly Asn Gly Gln Asp Ser Gln Asp Gly Gln Gly 565 570 575 Tyr Ser Ser Gly Arg Pro Gly Gln Gly Gly Arg Asn Gly Phe Gly Pro 580 585 590 Gly Gly Gln Asn Gly Asp Asn Asp Gly Ser Gly Tyr Arg Tyr 595 600 605 <210> 2 <211> 304 <212> PRT <213> Drosophila sechellia <400> 2 Arg Pro Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Ser Asp Ser Tyr 1 5 10 15 Gly Ala Pro Gly Gln Ser Gly Ala Gly Gly Arg Pro Ser Asp Thr Tyr 20 25 30 Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp Ser Tyr Gly 35 40 45 Ala Pro Gly Gln Gly Gln Gly Gln Gly Gln Gly Gln Gly Gly Tyr Gly 50 55 60 Gly Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn 65 70 75 80 Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly 85 90 95 Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly 100 105 110 Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Gly Asn Gly Asn 115 120 125 Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly 130 135 140 Asn Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly 145 150 155 160 Gly Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly 165 170 175 Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn 180 185 190 Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn 195 200 205 Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly 210 215 220 Asn Gly Ser Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ala Gln 225 230 235 240 Gly Gln Gly Gly Phe Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro 245 250 255 Gly Gln Asn Gln Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly 260 265 270 Asn Gly Ser Ala Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser 275 280 285 Gly Pro Gly Gly Arg Pro Ser Asp Ser Tyr Gly Pro Pro Ala Ser Gly 290 295 300 <210> 3 <211> 211 <212> PRT <213> Drosophila sechellia <400> 3 Gly Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn 1 5 10 15 Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly 20 25 30 Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly 35 40 45 Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Gly Asn Gly Asn 50 55 60 Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly 65 70 75 80 Asn Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly 85 90 95 Gly Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly 100 105 110 Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn 115 120 125 Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn 130 135 140 Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly 145 150 155 160 Asn Gly Ser Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ala Gln 165 170 175 Gly Gln Gly Gly Phe Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro 180 185 190 Gly Gln Asn Gln Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly 195 200 205 Asn Gly Ser 210 <210> 4 <211> 175 <212> PRT <213> Acromyrmex echinatior <400> 4 Phe Gly Glu Asn Arg Gly Asn Gly Gly Lys Pro Ser Thr Ser Tyr Gly 1 5 10 15 Val Pro Asp Ser Asn Gly Asn Asn Arg Gly Gly Phe Gly Asn Gly Gly 20 25 30 Ser Glu Gly Arg Pro Ser Thr Ser Tyr Gly Leu Pro Asp Ala Ser Arg 35 40 45 Asn Asn Gly Asn Gly Phe Gly Asn Val Gly Asn Glu Asp Lys Pro Ser 50 55 60 Thr Asn Tyr Gly Ile Pro Ala Asn Gly Asn Lys Val Ser Gly Phe Gly 65 70 75 80 Asn Val Gly Ser Glu Gly Arg Pro Ser Thr Ser Tyr Gly Val Pro Gly 85 90 95 Ala Asn Gly Asn Gln Gly Phe Gly Ser Gly Gly Ile Gly Gly Arg Pro 100 105 110 Ser Thr Ser Tyr Gly Val Pro Gly Val Asn Gly Asn Asn Gly Gly Gly 115 120 125 Phe Glu Asn Val Gly Arg Pro Ser Thr Ser Tyr Gly Thr Pro Asp Ala 130 135 140 Arg Gly Asn Asn Gly Gly Ser Phe Arg Asn Gly Asp Ile Gly Gly Arg 145 150 155 160 Pro Ser Thr Asn Tyr Gly Ile Pro Gly Ala Asn Gly Asn His Gly 165 170 175 <210> 5 <211> 158 <212> PRT <213> Aeshna sp. <400> 5 Ala Pro Ser Arg Gly Gly Gly His Gly Gly Gly Ser Ile Ser Ser Ser 1 5 10 15 Tyr Gly Ala Pro Ser Lys Gly Ser Gly Gly Phe Gly Gly Gly Ser Ile 20 25 30 Ser Ser Ser Tyr Gly Ala Pro Ser Lys Gly Ser Val Gly Gly Gly Val 35 40 45 Ser Ser Ser Tyr Gly Ala Pro Ala Ile Gly Gly Gly Ser Phe Gly Gly 50 55 60 Gly Ser Phe Gly Gly Gly Ser Phe Gly Gly Gly Ser Phe Gly Gly Gly 65 70 75 80 Ala Pro Ser Ser Ser Tyr Gly Ala Pro Ser Ser Ser Tyr Ser Ala Pro 85 90 95 Ser Ser Ser Tyr Gly Ala Pro Ser Lys Gly Ser Gly Gly Phe Gly Ser 100 105 110 Ser Gly Gly Phe Ser Ser Phe Ser Ser Ala Pro Ser Ser Ser Tyr Gly 115 120 125 Ala Pro Ser Ala Ser Tyr Ser Thr Pro Ser Ser Ser Tyr Gly Ala Pro 130 135 140 Ser Ser Gly Gly Phe Gly Ala Gly Gly Gly Phe Ser Ser Gly 145 150 155 <210> 6 <211> 457 <212> PRT <213> Aeshna sp. <400> 6 Glu Pro Pro Val Gly Gly Ser Gln Ser Tyr Leu Pro Pro Ser Ser Ser 1 5 10 15 Tyr Gly Ala Pro Ser Ala Gly Thr Gly Phe Gly His Gly Gly Gly Ser 20 25 30 Pro Ser Gln Ser Tyr Gly Ala Pro Ser Phe Gly Gly Gly Ser Val Gly 35 40 45 Gly Gly Ser His Phe Gly Gly Gly Ser His Ser Gly Gly Gly Gly Gly 50 55 60 Gly Tyr Pro Ser Gln Ser Tyr Gly Ala Pro Ser Arg Pro Ser Gly Ser 65 70 75 80 Ser Phe Gln Ala Phe Gly Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro 85 90 95 Ser Ser Gln Tyr Gly Ala Pro Ser Gly Gly Gly Gly Ser Tyr Ala Ile 100 105 110 Gln Gly Gly Ser Phe Ser Ser Gly Gly Ser Arg Ala Pro Ser Gln Ala 115 120 125 Tyr Gly Ala Pro Ser Asn Asn Ala Gly Leu Ser His Gln Ser Gln Ser 130 135 140 Phe Gly Gly Gly Leu Ser Ser Ser Tyr Gly Ala Pro Ser Ala Gly Phe 145 150 155 160 Gly Gly Gln Ser His Gly Gly Gly Tyr Ser Gln Gly Gly Asn Gly Gly 165 170 175 Gly His Gly Gly Ser Ser Gly Gly Gly Tyr Ser Tyr Gln Ser Phe Gly 180 185 190 Gly Gly Asn Gly Gly Gly His Gly Gly Ser Arg Pro Ser Ser Ser Tyr 195 200 205 Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro Ser Gly Gly Lys Gly Val 210 215 220 Ser Gly Gly Phe Val Ser Gln Pro Ser Gly Ser Tyr Gly Ala Pro Ser 225 230 235 240 Gln Ser Tyr Gly Ala Pro Ser Arg Gly Gly Gly His Gly Gly Gly Ser 245 250 255 Ile Ser Ser Ser Tyr Gly Ala Pro Ser Lys Gly Ser Gly Gly Phe Gly 260 265 270 Gly Gly Ser Ile Ser Ser Ser Tyr Gly Ala Pro Ser Lys Gly Ser Val 275 280 285 Gly Gly Gly Val Ser Ser Ser Tyr Gly Ala Pro Ala Ile Gly Gly Gly 290 295 300 Ser Phe Gly Gly Gly Ser Phe Gly Gly Gly Ser Phe Gly Gly Gly Ser 305 310 315 320 Phe Gly Gly Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro Ser Ser Ser 325 330 335 Tyr Ser Ala Pro Ser Ser Ser Tyr Gly Ala Pro Ser Lys Gly Ser Gly 340 345 350 Gly Phe Gly Ser Ser Gly Gly Phe Ser Ser Phe Ser Ser Ala Pro Ser 355 360 365 Ser Ser Tyr Gly Ala Pro Ser Ala Ser Tyr Ser Thr Pro Ser Ser Ser 370 375 380 Tyr Gly Ala Pro Ser Ser Gly Gly Phe Gly Ala Gly Gly Gly Phe Ser 385 390 395 400 Ser Gly Gly Tyr Ser Gly Gly Gly Gly Gly Tyr Ser Ser Gly Gly Ser 405 410 415 Gly Gly Phe Gly Gly His Gly Gly Ser Gly Gly Ala Gly Gly Tyr Ser 420 425 430 Gly Gly Gly Gly Tyr Ser Gly Gly Gly Ser Gly Gly Gly Gln Lys Tyr 435 440 445 Asp Ser Asn Gly Gly Tyr Val Tyr Ser 450 455 <210> 7 <211> 162 <212> PRT <213> Haematobia irritans <400> 7 Ala Gly Gly Gly Asn Gly Gly Gly Gly Thr Gly Gly Thr Pro Ser Ser 1 5 10 15 Ser Tyr Gly Ala Pro Ser Asn Gly Gly Gly Ser Asn Gly Asn Gly Phe 20 25 30 Gly Ser Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Gly Gly Ser Asn 35 40 45 Gly Asn Gly Gly Gly Arg Pro Ser Leu Ser Tyr Gly Ala Pro Gly Ser 50 55 60 Gly Gly Ser Asn Gly Asn Gly Gly Gly Arg Pro Ser Ser Ser Tyr Gly 65 70 75 80 Ala Pro Gly Ala Gly Gly Ser Asn Gly Asn Gly Gly Gly Arg Pro Ser 85 90 95 Ser Ser Tyr Gly Ala Pro Gly Ala Gly Gly Ser Asn Gly Asn Gly Gly 100 105 110 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ala Gly Gly Ser Asn 115 120 125 Gly Asn Gly Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ala 130 135 140 Gly Gly Ser Asn Gly Asn Gly Gly Ser Arg Pro Ser Ser Thr Tyr Gly 145 150 155 160 Ala Pro <210> 8 <211> 580 <212> PRT <213> Haematobia irritans <400> 8 Arg Pro Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Pro Leu Asn Asn 1 5 10 15 Tyr Gly Ala Pro Gly Ala Gly Gly Gly Ser Ser Asp Gly Ser Pro Leu 20 25 30 Ala Pro Ser Asp Ala Tyr Gly Ala Pro Asp Leu Gly Gly Gly Ser Gly 35 40 45 Gly Ser Gly Gln Gly Pro Ser Ser Ser Tyr Gly Ala Pro Gly Leu Gly 50 55 60 Gly Gly Asn Gly Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro Gly Leu 65 70 75 80 Gly Gly Gly Asn Gly Gly Ser Arg Arg Pro Ser Ser Ser Tyr Gly Ala 85 90 95 Pro Gly Ala Gly Gly Gly Asn Gly Gly Gly Gly Thr Gly Gly Thr Pro 100 105 110 Ser Ser Ser Tyr Gly Ala Pro Ser Asn Gly Gly Gly Ser Asn Gly Asn 115 120 125 Gly Phe Gly Ser Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Gly Gly 130 135 140 Ser Asn Gly Asn Gly Gly Gly Arg Pro Ser Leu Ser Tyr Gly Ala Pro 145 150 155 160 Gly Ser Gly Gly Ser Asn Gly Asn Gly Gly Gly Arg Pro Ser Ser Ser 165 170 175 Tyr Gly Ala Pro Gly Ala Gly Gly Ser Asn Gly Asn Gly Gly Gly Arg 180 185 190 Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ala Gly Gly Ser Asn Gly Asn 195 200 205 Gly Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ala Gly Gly 210 215 220 Ser Asn Gly Asn Gly Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro 225 230 235 240 Gly Ala Gly Gly Ser Asn Gly Asn Gly Gly Ser Arg Pro Ser Ser Thr 245 250 255 Tyr Gly Ala Pro Gly Ala Gly Gly Ser Asn Gly Asn Gly Cys Gly Asn 260 265 270 Lys Pro Ser Ser Ser Tyr Gly Ala Pro Ser Ala Gly Ser Asn Gly Asn 275 280 285 Gly Gly Ser Glu Gln Gly Ser Ser Gly Ser Pro Ser Asp Ser Tyr Gly 290 295 300 Pro Pro Ala Ser Gly Thr Gly Arg Gly Arg Asn Gly Gly Gly Gly Gly 305 310 315 320 Ala Gly Gly Gly Arg Arg Gly Gln Pro Asn Gln Glu Tyr Leu Pro Pro 325 330 335 Asn Gln Gly Asp Asn Gly Asn Asn Gly Gly Ser Gly Gly Asp Asp Gly 340 345 350 Tyr Asp Tyr Ser Gln Ser Gly Asp Gly Gly Gly Gln Gly Gly Ser Gly 355 360 365 Gly Ser Gly Asn Gly Gly Asp Asp Gly Ser Asn Ile Val Glu Tyr Glu 370 375 380 Ala Gly Gln Glu Gly Tyr Arg Pro Gln Ile Arg Tyr Glu Gly Glu Ala 385 390 395 400 Asn Glu Gly Gly Gln Gly Ser Gly Gly Ala Gly Gly Ser Asp Gly Thr 405 410 415 Asp Gly Tyr Glu Tyr Glu Gln Asn Gly Gly Asp Gly Gly Ala Gly Gly 420 425 430 Ser Gly Gly Pro Gly Thr Gly Gln Asp Leu Gly Glu Asn Gly Tyr Ser 435 440 445 Ser Gly Arg Pro Gly Gly Asp Asn Gly Gly Gly Gly Gly Tyr Ser Asn 450 455 460 Gly Asn Gly Gln Gly Asp Gly Gly Gln Asp Leu Gly Ser Asn Gly Tyr 465 470 475 480 Ser Ser Gly Ala Pro Asn Gly Gln Asn Gly Gly Arg Arg Asn Gly Gly 485 490 495 Gly Gln Asn Asn Asn Gly Gln Gly Tyr Ser Ser Gly Arg Pro Asn Gly 500 505 510 Asn Gly Ser Gly Gly Arg Asn Gly Asn Gly Gly Arg Gly Asn Gly Gly 515 520 525 Gly Tyr Arg Asn Gly Asn Gly Asn Gly Gly Gly Asn Gly Asn Gly Ser 530 535 540 Gly Ser Gly Ser Gly Asn Asn Gly Tyr Asn Tyr Asp Gln Gln Gly Ser 545 550 555 560 Asn Gly Phe Gly Ala Gly Gly Gln Asn Gly Glu Asn Asp Gly Ser Gly 565 570 575 Tyr Arg Tyr Ser 580 <210> 9 <211> 256 <212> PRT <213> Ctenocephalides felis <400> 9 Ala Asn Gly Asn Gly Phe Glu Gly Ala Ser Asn Gly Leu Ser Ala Thr 1 5 10 15 Tyr Gly Ala Pro Asn Gly Gly Gly Phe Gly Gly Asn Gly Asn Gly Gly 20 25 30 Ala Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ala Gly Asn Gly Gly Asn 35 40 45 Gly Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ala Gly Gly 50 55 60 Ser Gly Asn Gly Phe Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro 65 70 75 80 Gly Asn Gly Asn Gly Ala Asn Gly Gly Arg Gly Gly Arg Pro Ser Ser 85 90 95 Arg Tyr Gly Ala Pro Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly 100 105 110 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Asn Gly Asn Gly 115 120 125 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Gly Asn Gly Phe 130 135 140 Gly Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ala 145 150 155 160 Asn Gly Asn Gly Asn Gly Gly Ala Ile Gly Gln Pro Ser Ser Ser Tyr 165 170 175 Gly Ala Pro Gly Gln Asn Gly Asn Gly Gly Gly Leu Ser Ser Thr Tyr 180 185 190 Gly Ala Pro Gly Ala Gly Asn Gly Gly Phe Gly Gly Asn Gly Gly Gly 195 200 205 Leu Ser Ser Thr Tyr Gly Ala Pro Gly Ser Gly Asn Gly Gly Phe Gly 210 215 220 Gly Asn Gly Leu Ser Ser Thr Tyr Gly Ala Pro Gly Ser Gly Asn Gly 225 230 235 240 Gly Phe Gly Gly Asn Gly Gly Gly Leu Ser Ser Thr Tyr Gly Ala Pro 245 250 255 <210> 10 <211> 146 <212> PRT <213> Ctenocephalides felis <400> 10 Pro Gly Gly Ala Gly Gly Ala Gly Gly Tyr Pro Gly Gly Ala Gly Gly 1 5 10 15 Ala Gly Gly Ala Gly Gly Tyr Pro Gly Gly Ser Ala Gly Gly Ala Gly 20 25 30 Gly Tyr Pro Gly Gly Ser Gly Ser Gly Val Gly Gly Tyr Pro Gly Gly 35 40 45 Ser Asn Gly Gly Ala Gly Gly Tyr Pro Gly Gly Ser Asn Gly Gly Ala 50 55 60 Gly Gly Tyr Pro Gly Gly Ser Asn Gly Gly Ala Gly Gly Tyr Pro Gly 65 70 75 80 Gly Ser Asn Gly Gly Ala Gly Gly Tyr Pro Gly Gly Ser Asn Gly Asn 85 90 95 Gly Gly Tyr Ser Asn Gly Gly Ser Asn Gly Gly Gly Ala Gly Gly Tyr 100 105 110 Pro Gly Gly Ser Asn Gly Asn Gly Gly Tyr Pro Gly Ser Gly Ser Asn 115 120 125 Gly Gly Ala Gly Gly Tyr Pro Gly Gly Ser Asn Gly Asn Gly Gly Tyr 130 135 140 Pro Gly 145 <210> 11 <211> 201 <212> PRT <213> Bombus terrestris <400> 11 Phe Asp Gly Gln Asn Gly Ile Gly Gly Gly Asp Ser Gly Arg Asn Gly 1 5 10 15 Leu Ser Asn Ser Tyr Gly Val Pro Gly Ser Asn Gly Gly Arg Asn Gly 20 25 30 Asn Gly Arg Gly Asn Gly Phe Gly Gly Gly Gln Pro Ser Ser Ser Tyr 35 40 45 Gly Ala Pro Ser Asn Gly Leu Gly Gly Asn Gly Gly Ser Gly Ala Gly 50 55 60 Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Phe Gly 65 70 75 80 Gly Gly Gln Pro Ser Ser Ser Tyr Gly Ala Pro Ser Asn Gly Leu Gly 85 90 95 Gly Asn Gly Ala Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly 100 105 110 Gly Asn Gly Phe Gly Gly Gly Ser Asn Gly Ala Gly Lys Asn Gly Phe 115 120 125 Gly Gly Ala Pro Ser Asn Ser Tyr Gly Pro Pro Glu Asn Gly Asn Gly 130 135 140 Phe Gly Gly Gly Asn Gly Gly Gly Ser Pro Ser Gly Leu Tyr Gly Pro 145 150 155 160 Pro Gly Arg Asn Gly Gly Asn Gly Gly Asn Gly Gly Asn Gly Gly Asn 165 170 175 Gly Gly Arg Pro Ser Ser Ser Tyr Gly Thr Pro Glu Arg Asn Gly Gly 180 185 190 Arg Pro Ser Gly Leu Tyr Gly Pro Pro 195 200 <210> 12 <211> 183 <212> PRT <213> Tribolium castaneum <400> 12 Asn Gly Phe Gly Gly Gly Gln Asn Gly Gly Arg Leu Ser Ser Thr Tyr 1 5 10 15 Gly Pro Pro Gly Gln Gly Gly Asn Gly Phe Gly Gly Gly Gln Asn Gly 20 25 30 Gly Arg Pro Ser Ser Thr Tyr Gly Pro Pro Gly Gln Gly Gly Asn Gly 35 40 45 Phe Gly Gly Gly Gln Asn Gly Gly Arg Pro Ser Ser Thr Tyr Gly Pro 50 55 60 Pro Gly Gln Gly Gly Asn Gly Phe Gly Gly Gly Gln Asn Gly Gly Arg 65 70 75 80 Pro Ser Ser Thr Tyr Gly Pro Pro Gly Gln Gly Gly Asn Gly Phe Gly 85 90 95 Gly Gly Gln Asn Gly Gly Arg Pro Ser Ser Thr Tyr Gly Pro Pro Gly 100 105 110 Gln Gly Gly Asn Gly Phe Gly Gly Gly Gln Asn Gly Gly Arg Pro Ser 115 120 125 Ser Thr Tyr Gly Pro Pro Gly Gln Gly Gly Asn Gly Phe Gly Gly Gly 130 135 140 Gln Asn Gly Gly Lys Pro Ser Ser Thr Tyr Gly Pro Pro Gly Gln Gly 145 150 155 160 Gly Asn Gly Phe Gly Gly Gly Gln Asn Gly Gly Arg Pro Ser Ser Thr 165 170 175 Tyr Gly Pro Pro Gly Gln Gly 180 <210> 13 <211> 551 <212> PRT <213> Tribolium castaneum <400> 13 Arg Ala Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Ser Gln Asn Gly 1 5 10 15 Gly Pro Ser Ser Thr Tyr Gly Pro Pro Gly Phe Gln Pro Gly Thr Pro 20 25 30 Leu Gly Gly Gly Gly Asn Gly Gly His Pro Pro Ser Gln Gly Gly Asn 35 40 45 Gly Gly Phe Gly Gly Arg His Pro Asp Ser Asp Gln Arg Pro Gly Thr 50 55 60 Ser Tyr Leu Pro Pro Gly Gln Asn Gly Gly Ala Gly Arg Pro Gly Val 65 70 75 80 Thr Tyr Gly Pro Pro Gly Gln Gly Gly Gly Gln Asn Gly Gly Gly Pro 85 90 95 Ser Ser Thr Tyr Gly Pro Pro Gly Gln Gly Gly Asn Gly Phe Gly Gly 100 105 110 Gly Gln Asn Gly Gly Arg Leu Ser Ser Thr Tyr Gly Pro Pro Gly Gln 115 120 125 Gly Gly Asn Gly Phe Gly Gly Gly Gln Asn Gly Gly Arg Pro Ser Ser 130 135 140 Thr Tyr Gly Pro Pro Gly Gln Gly Gly Asn Gly Phe Gly Gly Gly Gln 145 150 155 160 Asn Gly Gly Arg Pro Ser Ser Thr Tyr Gly Pro Pro Gly Gln Gly Gly 165 170 175 Asn Gly Phe Gly Gly Gly Gln Asn Gly Gly Arg Pro Ser Ser Thr Tyr 180 185 190 Gly Pro Pro Gly Gln Gly Gly Asn Gly Phe Gly Gly Gly Gln Asn Gly 195 200 205 Gly Arg Pro Ser Ser Thr Tyr Gly Pro Pro Gly Gln Gly Gly Asn Gly 210 215 220 Phe Gly Gly Gly Gln Asn Gly Gly Arg Pro Ser Ser Thr Tyr Gly Pro 225 230 235 240 Pro Gly Gln Gly Gly Asn Gly Phe Gly Gly Gly Gln Asn Gly Gly Lys 245 250 255 Pro Ser Ser Thr Tyr Gly Pro Pro Gly Gln Gly Gly Asn Gly Phe Gly 260 265 270 Gly Gly Gln Asn Gly Gly Arg Pro Ser Ser Thr Tyr Gly Pro Pro Gly 275 280 285 Gln Gly Gly Asn Gly Asn Gly Gly Gly His Asn Gly Gln Arg Pro Gly 290 295 300 Gly Ser Tyr Leu Pro Pro Ser Gln Gly Gly Asn Gly Gly Tyr Pro Ser 305 310 315 320 Gly Gly Pro Gly Gly Tyr Pro Ser Gly Gly Pro Gly Gly Asn Gly Gly 325 330 335 Tyr Gly Gly Glu Glu Glu Ser Thr Glu Pro Ala Lys Tyr Glu Phe Glu 340 345 350 Tyr Gln Val Asp Asp Asp Glu His Asn Thr His Phe Gly His Gln Glu 355 360 365 Ser Arg Asp Gly Asp Lys Ala Thr Gly Glu Tyr Asn Val Leu Leu Pro 370 375 380 Asp Gly Arg Lys Gln Val Val Gln Tyr Glu Ala Asp Ser Glu Gly Tyr 385 390 395 400 Lys Pro Lys Ile Ser Tyr Glu Gly Gly Asn Gly Asn Gly Gly Tyr Pro 405 410 415 Ser Gly Gly Pro Gly Gly Ala Gly Asn Gly Gly Tyr Pro Ser Gly Gly 420 425 430 Pro Gln Gly Gly Asn Gly Gly Tyr Pro Ser Gly Gly Pro Gln Gly Gly 435 440 445 Asn Gly Gly Tyr Pro Ser Gly Gly Pro Gln Gly Gly Asn Gly Gly Tyr 450 455 460 Pro Ser Gly Gly Pro Gln Gly Gly Asn Gly Gly Tyr Pro Ser Gly Gly 465 470 475 480 Pro Gln Gly Gly Asn Gly Gly Tyr Pro Ser Gly Gly Pro Gln Gly Gly 485 490 495 Asn Gly Gly Tyr Pro Ser Gly Gly Pro Gln Gly Gly Asn Gly Gly Tyr 500 505 510 Pro Ser Gly Gly Pro Gln Gly Gly Asn Gly Gly Tyr Thr Ser Gly Gly 515 520 525 Pro Gln Gly Gly Asn Gly Gly Tyr Pro Ser Gly Gly Pro Gln Gly Gly 530 535 540 Asn Gly Gly Ser Gly Pro Tyr 545 550 <210> 14 <211> 444 <212> PRT <213> Tribolium castaneum <400> 14 Gln Leu Thr Lys Arg Asp Ala Pro Leu Ser Gly Gly Tyr Pro Ser Gly 1 5 10 15 Gly Pro Ala Asn Ser Tyr Leu Pro Pro Gly Gly Ala Ser Gln Pro Ser 20 25 30 Gly Asn Tyr Gly Ala Pro Ser Gly Gly Phe Gly Gly Lys Ser Gly Gly 35 40 45 Phe Gly Gly Ser Gly Gly Phe Gly Gly Ala Pro Ser Gln Ser Tyr Gly 50 55 60 Ala Pro Ser Gly Gly Phe Gly Gly Ser Ser Ser Phe Gly Lys Ser Gly 65 70 75 80 Gly Phe Gly Gly Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Gly Gly 85 90 95 Phe Gly Gly Ser Ser Ser Phe Gly Lys Ser Ser Gly Gly Phe Gly Gly 100 105 110 Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Gly Gly Phe Gly Gly Ser 115 120 125 Ser Ser Phe Gly Lys Ser Gly Gly Phe Gly Gly Ala Pro Ser Gln Ser 130 135 140 Tyr Gly Ala Pro Ser Gly Gly Phe Gly Gly Ser Ser Ser Phe Gly Lys 145 150 155 160 Ser Gly Gly Phe Gly Gly Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser 165 170 175 Gly Gly Phe Gly Gly Lys Ser Ser Ser Phe Ser Ser Ala Pro Ser Gln 180 185 190 Ser Tyr Gly Ala Pro Ser Gly Gly Phe Gly Gly Lys Ser Gly Gly Phe 195 200 205 Gly Gly Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Gly Gly Phe Gly 210 215 220 Gly Lys Ser Gly Gly Phe Gly Gly Ala Pro Ser Gln Ser Tyr Gly Ala 225 230 235 240 Pro Ser Gly Gly Phe Gly Gly Ser Ser Ser Phe Gly Lys Ser Gly Gly 245 250 255 Phe Gly Gly Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Gly Gly Phe 260 265 270 Gly Gly Ser Ser Ser Phe Gly Lys Ser Ser Gly Phe Gly His Gly Ser 275 280 285 Gly Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Arg Ser Gln Pro Gln 290 295 300 Ser Asn Tyr Leu Pro Pro Ser Thr Ser Tyr Gly Thr Pro Val Ser Ser 305 310 315 320 Ala Lys Ser Ser Gly Ser Phe Gly Gly Ala Pro Ser Gln Ser Tyr Gly 325 330 335 Ala Pro Ser Gln Ser His Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser 340 345 350 Arg Ser Phe Ser Gln Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Gln 355 360 365 Gly His Ala Pro Ala Pro Gln Gln Ser Tyr Ser Ala Pro Ser Gln Ser 370 375 380 Tyr Gly Ala Pro Ser Gly Gly Phe Gly Gly Gly His Gly Gly Phe Gly 385 390 395 400 Gly Gln Gly Gln Gly Phe Gly Gly Gly Arg Ser Gln Pro Ser Gln Ser 405 410 415 Tyr Gly Ala Pro Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Ala Gly 420 425 430 Gly Gln Gln Tyr Ala Ser Asn Gly Gly Tyr Ser Tyr 435 440 <210> 15 <211> 426 <212> PRT <213> Apis mellifera <400> 15 Arg Ser Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Ser Gly Asn Gly 1 5 10 15 Asn Gly Gly Gly Gly Gly Gly Ser Ser Asn Val Tyr Gly Pro Pro Gly 20 25 30 Phe Asp Gly Gln Asn Gly Ile Gly Glu Gly Asp Asn Gly Arg Asn Gly 35 40 45 Ile Ser Asn Ser Tyr Gly Val Pro Thr Gly Gly Asn Gly Tyr Asn Gly 50 55 60 Asp Ser Ser Gly Asn Gly Arg Pro Gly Thr Asn Gly Gly Arg Asn Gly 65 70 75 80 Asn Gly Asn Gly Arg Gly Asn Gly Tyr Gly Gly Gly Gln Pro Ser Asn 85 90 95 Ser Tyr Gly Pro Pro Ser Asn Gly His Gly Gly Asn Gly Ala Gly Arg 100 105 110 Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Phe Ala Gly 115 120 125 Gly Ser Asn Gly Lys Asn Gly Phe Gly Gly Gly Pro Ser Ser Ser Tyr 130 135 140 Gly Pro Pro Glu Asn Gly Asn Gly Phe Asn Gly Gly Asn Gly Gly Pro 145 150 155 160 Ser Gly Leu Tyr Gly Pro Pro Gly Arg Asn Gly Gly Asn Gly Gly Asn 165 170 175 Gly Gly Asn Gly Gly Arg Pro Ser Gly Ser Tyr Gly Thr Pro Glu Arg 180 185 190 Asn Gly Gly Arg Leu Gly Gly Leu Tyr Gly Ala Pro Gly Arg Asn Gly 195 200 205 Asn Asn Gly Gly Asn Gly Tyr Pro Ser Gly Gly Leu Asn Gly Gly Asn 210 215 220 Gly Gly Tyr Pro Ser Gly Gly Pro Gly Asn Gly Gly Ala Asn Gly Gly 225 230 235 240 Tyr Pro Ser Gly Gly Ser Asn Gly Asp Asn Gly Gly Tyr Pro Ser Gly 245 250 255 Gly Pro Asn Gly Asn Gly Asn Gly Asn Gly Gly Tyr Gly Gln Asp Glu 260 265 270 Asn Asn Glu Pro Ala Lys Tyr Glu Phe Ser Tyr Glu Val Lys Asp Glu 275 280 285 Gln Ser Gly Ala Asp Tyr Gly His Thr Glu Ser Arg Asp Gly Asp Arg 290 295 300 Ala Gln Gly Glu Phe Asn Val Leu Leu Pro Asp Gly Arg Lys Gln Ile 305 310 315 320 Val Glu Tyr Glu Ala Asp Gln Asp Gly Phe Lys Pro Gln Ile Arg Tyr 325 330 335 Glu Gly Glu Ala Asn Ser Gln Gly Tyr Gly Ser Gly Gly Pro Gly Gly 340 345 350 Asn Gly Gly Asp Asn Gly Tyr Pro Ser Gly Gly Pro Gly Gly Asn Gly 355 360 365 Tyr Ser Ser Gly Arg Pro Asn Gly Gly Ser Asp Phe Ser Asp Gly Gly 370 375 380 Tyr Pro Ser Thr Arg Pro Gly Gly Glu Asn Gly Gly Tyr Arg Asn Gly 385 390 395 400 Asn Asn Gly Gly Asn Gly Asn Gly Gly Tyr Pro Ser Gly Asn Gly Gly 405 410 415 Asp Ala Ala Ala Asn Gly Gly Tyr Gln Tyr 420 425 <210> 16 <211> 318 <212> PRT <213> Apis mellifera <400> 16 Asp Ala Pro Ile Ser Gly Ser Tyr Leu Pro Pro Ser Thr Ser Tyr Gly 1 5 10 15 Thr Pro Asn Leu Gly Gly Gly Gly Pro Ser Ser Thr Tyr Gly Ala Pro 20 25 30 Ser Gly Gly Gly Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ser 35 40 45 Ser Thr Tyr Gly Ala Pro Ser Ser Thr Tyr Gly Ala Pro Ser Asn Gly 50 55 60 Gly Gly Arg Pro Ser Ser Thr Tyr Gly Ala Pro Ser Asn Gly Gly Gly 65 70 75 80 Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro 85 90 95 Ser Ser Thr Tyr Gly Ala Pro Ser Asn Gly Gly Gly Arg Pro Ser Ser 100 105 110 Ser Tyr Gly Ala Pro Ser Phe Gly Gly Gly Gly Gly Phe Gly Gly Gly 115 120 125 Asn Gly Leu Ser Thr Ser Tyr Gly Ala Pro Ser Arg Gly Gly Gly Gly 130 135 140 Gly Gly Gly Ser Ile Ser Ser Ser Tyr Gly Ala Pro Thr Gly Gly Gly 145 150 155 160 Gly Gly Gly Pro Ser Thr Thr Tyr Gly Ala Pro Asn Gly Gly Gly Asn 165 170 175 Gly Tyr Ser Arg Pro Ser Ser Thr Tyr Gly Thr Pro Ser Thr Gly Gly 180 185 190 Gly Ser Phe Gly Gly Ser Gly Gly Tyr Ser Gly Gly Gly Gly Gly Tyr 195 200 205 Ser Gly Gly Gly Asn Gly Tyr Ser Gly Gly Gly Gly Gly Gly Tyr Ser 210 215 220 Gly Gly Asn Gly Gly Gly Tyr Ser Gly Gly Gly Asn Gly Gly Gly Tyr 225 230 235 240 Ser Gly Gly Asn Gly Gly Gly Tyr Ser Gly Gly Gly Gly Gly Gly Tyr 245 250 255 Ser Gly Gly Gly Gly Gly Gly Tyr Ser Gly Gly Gly Asn Gly Tyr Ser 260 265 270 Gly Gly Gly Gly Gly Gly Tyr Ser Gly Gly Asn Gly Gly Tyr Ser Gly 275 280 285 Gly Asn Gly Gly Tyr Ser Gly Gly Gly Gly Gly Tyr Ser Gly Gly Gly 290 295 300 Gly Gly Gly Gln Ser Tyr Ala Ser Asn Gly Gly Tyr Gln Tyr 305 310 315 <210> 17 <211> 747 <212> PRT <213> Nasonia vitripennis <400> 17 Arg Pro Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Gly Gln Gly Gly 1 5 10 15 Gly Phe Gly Gly Gly Arg Pro Ser Gly Ala Ser Pro Ser Asp Gln Tyr 20 25 30 Gly Pro Pro Asp Phe Gln Gly Ala Gly Gly Arg Gly Gly Gln Ala Ala 35 40 45 Gly Gly Asn Phe Gly Gly Gly Gly Asn Gly Phe Gly Gly Ala Pro Ser 50 55 60 Ser Ser Tyr Gly Pro Pro Gly Phe Gly Ser Asn Glu Pro Asn Lys Phe 65 70 75 80 Ser Gly Ala Gly Gly Gly Gly Ala Gly Arg Pro Gln Asp Ser Tyr Gly 85 90 95 Pro Pro Ala Gly Gly Asn Gly Phe Ala Gly Ser Ala Gly Ala Gly Asn 100 105 110 Ser Gly Arg Pro Gly Gly Ala Ala Ala Gly Gly Arg Pro Ser Asp Ser 115 120 125 Tyr Gly Pro Pro Gln Gly Gly Gly Ser Gly Phe Gly Gly Gly Asn Ala 130 135 140 Gly Arg Pro Ser Asp Ser Tyr Gly Pro Pro Ser Ala Gly Gly Gly Gly 145 150 155 160 Phe Gly Gly Gly Ser Pro Gly Gly Gly Phe Gly Gly Gly Ser Pro Gly 165 170 175 Gly Gly Phe Gly Gly Gly Asn Gln Gly Ala Pro Gln Ser Ser Tyr Gly 180 185 190 Pro Pro Ala Ser Gly Phe Gly Gly Gln Gly Gly Ala Gly Gln Gly Arg 195 200 205 Pro Ser Asp Ser Tyr Gly Pro Pro Gly Gly Gly Ser Gly Gly Arg Pro 210 215 220 Ser Gln Gly Gly Asn Gly Phe Gly Gly Gly Asn Ala Gly Arg Pro Ser 225 230 235 240 Asp Ser Tyr Gly Pro Pro Ala Ala Gly Gly Gly Gly Phe Gly Gly Asn 245 250 255 Ala Gly Gly Asn Gly Gly Gly Asn Gly Phe Gly Gly Gly Arg Pro Ser 260 265 270 Gly Ser Pro Gly Gly Phe Gly Gly Gln Gly Gly Gly Gly Arg Pro Ser 275 280 285 Asp Ser Tyr Leu Pro Pro Ser Gly Gly Ser Gly Phe Gly Gly Gly Asn 290 295 300 Gly Arg Gln Pro Gly Gly Phe Gly Gln Gln Gly Gly Asn Gly Ala Gly 305 310 315 320 Gln Gln Asn Gly Gly Gly Gly Ala Gly Arg Pro Ser Ser Ser Tyr Gly 325 330 335 Pro Pro Ser Asn Gly Asn Gly Gly Gly Phe Ser Gly Gln Asn Gly Gly 340 345 350 Arg Gly Ser Pro Ser Ser Gly Gly Gly Phe Gly Gly Ala Gly Gly Ser 355 360 365 Pro Ser Ser Ser Tyr Gly Pro Pro Ala Gly Gly Ser Gly Phe Gly Asn 370 375 380 Asn Gly Gly Ala Gly Gly Arg Pro Ser Ser Ser Tyr Gly Pro Pro Ser 385 390 395 400 Ser Gly Gly Asn Gly Phe Gly Ser Gly Gly Gln Gly Gly Gln Gly Gly 405 410 415 Gln Gly Gly Gln Gly Gly Arg Pro Ser Ser Ser Tyr Gly Pro Pro Ser 420 425 430 Asn Gly Asn Gly Gly Phe Gly Gly Gly Asn Gly Gly Arg Pro Ser Ser 435 440 445 Asn Gly Tyr Pro Gln Gly Gln Gly Asn Gly Asn Gly Gly Phe Gly Gly 450 455 460 Gln Gly Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Pro Pro Gly 465 470 475 480 Gly Asp Ser Gly Tyr Pro Ser Gly Gly Pro Ser Gly Asn Phe Gly Gly 485 490 495 Ser Asn Ala Gly Gly Gly Gly Gly Gly Phe Gly Gly Gln Val Gln Asp 500 505 510 Ser Tyr Gly Pro Pro Pro Ser Gly Ala Val Asn Gly Asn Gly Asn Gly 515 520 525 Tyr Ser Ser Gly Gly Pro Gly Gly Asn Gly Leu Asp Glu Gly Asn Asp 530 535 540 Glu Pro Ala Lys Tyr Glu Phe Ser Tyr Glu Val Lys Asp Asp Gln Ser 545 550 555 560 Asp Gly Arg Lys Gln Ile Val Glu Tyr Glu Ala Asp Gln Asp Gly Phe 565 570 575 Lys Pro Gln Ile Arg Tyr Glu Gly Glu Ala Asn Thr Gly Ala Gly Gly 580 585 590 Ala Gly Gly Tyr Pro Ser Gly Gly Gly Gly Asp Ser Gly Tyr Pro Ser 595 600 605 Gly Pro Ser Gly Ala Gly Gly Asn Ala Gly Tyr Pro Ser Gly Gly Gly 610 615 620 Gly Gly Ala Gly Gly Phe Gly Gly Asn Gly Gly Gly Ser Asn Gly Tyr 625 630 635 640 Pro Ser Gly Gly Pro Ser Gly Gly Gln Gly Gln Phe Gly Gly Gln Gln 645 650 655 Gly Gly Asn Gly Gly Tyr Pro Ser Gly Pro Gln Gly Gly Ser Gly Phe 660 665 670 Gly Gly Gly Ser Gln Gly Ser Gly Ser Gly Gly Tyr Pro Ser Gly Gly 675 680 685 Pro Gly Gly Asn Gly Gly Asn Asn Asn Phe Gly Gly Gly Asn Ala Gly 690 695 700 Tyr Pro Ser Gly Gly Pro Ser Gly Gly Asn Gly Phe Asn Gln Gly Gly 705 710 715 720 Gln Asn Gln Gly Gly Ser Gly Gly Gly Tyr Pro Ser Gly Ser Gly Gly 725 730 735 Asp Ala Ala Ala Asn Gly Gly Tyr Gln Tyr Ser 740 745 <210> 18 <211> 419 <212> PRT <213> Nasonia vitripennis <400> 18 Arg Ala Glu Ala Pro Ile Ser Gly Asn Tyr Leu Pro Pro Ser Thr Ser 1 5 10 15 Tyr Gly Thr Pro Asn Leu Gly Gly Gly Gly Gly Gly Gly Gly Gly Phe 20 25 30 Gly Gly Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro Ser Ser Gly Gly 35 40 45 Gly Phe Gly Gly Ser Phe Gly Gly Gly Ala Pro Ser Ser Ser Tyr Gly 50 55 60 Ala Pro Ser Thr Gly Gly Ser Phe Gly Gly Gly Ala Pro Ser Ser Ser 65 70 75 80 Tyr Gly Ala Pro Ser Ser Gly Gly Ser Phe Gly Gly Ser Phe Gly Gly 85 90 95 Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro Ser Phe Gly Gly Asn Ala 100 105 110 Pro Ser Ser Ser Tyr Gly Ala Pro Ser Ala Gly Gly Ser Phe Gly Gly 115 120 125 Gly Ala Pro Ser Asn Ser Tyr Gly Pro Pro Ser Ser Ser Tyr Gly Ala 130 135 140 Pro Ser Ala Gly Gly Ser Phe Gly Gly Ser Ser Gly Gly Ser Phe Gly 145 150 155 160 Gly Ser Phe Gly Gly Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro Ala 165 170 175 Pro Ser Arg Pro Ser Ser Asn Tyr Gly Ala Pro Ser Arg Pro Ser Ser 180 185 190 Asn Tyr Gly Ala Pro Ser Ser Gly Gly Ser Gly Phe Gly Gly Gly Ser 195 200 205 Gly Phe Gly Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ser Ser 210 215 220 Gly Ser Phe Gly Gly Gly Phe Gly Gly Gly Ala Pro Ser Ser Ser Tyr 225 230 235 240 Gly Ala Pro Ala Pro Ser Arg Pro Ser Ser Asn Tyr Gly Ala Pro Ala 245 250 255 Pro Ser Arg Pro Ser Ser Asn Tyr Gly Ala Pro Ala Pro Ser Arg Pro 260 265 270 Ser Ser Ser Tyr Gly Ala Pro Ser Arg Pro Ser Ser Asn Tyr Gly Ala 275 280 285 Pro Ser Arg Pro Ser Ser Asn Tyr Gly Ala Pro Ser Ser Gly Gly Ser 290 295 300 Gly Phe Gly Gly Gly Ser Gly Phe Gly Gly Gly Arg Pro Ser Ser Ser 305 310 315 320 Tyr Gly Ala Pro Ser Ser Gly Ser Phe Gly Gly Gly Phe Gly Gly Gly 325 330 335 Ala Pro Ser Ser Ser Tyr Gly Ala Pro Ala Pro Ser Arg Pro Ser Ser 340 345 350 Asn Tyr Gly Pro Pro Ser Ser Ser Tyr Gly Ala Pro Ser Ser Gly Gly 355 360 365 Ser Gly Gly Phe Gly Gly Gly Ala Pro Ser Ser Ser Tyr Gly Ala Pro 370 375 380 Ser Phe Gly Gly Ser Ser Asn Ala Val Ser Arg Pro Ser Ser Ser Tyr 385 390 395 400 Gly Ala Pro Ser Ser Gly Gly Gly Gln Ser Tyr Ala Ser Asn Gly Gly 405 410 415 Tyr Gln Tyr <210> 19 <211> 396 <212> PRT <213> Human louse <400> 19 Glu Pro Pro Val Lys Thr Ser Tyr Leu Pro Pro Ser Ala Ser Arg Ser 1 5 10 15 Leu Asn Ser Gln Tyr Gly Ala Pro Ala Phe Thr Asp Ser Asn Glu Leu 20 25 30 Val Ala Pro Ser Pro Asn Ser Asn Phe His Asp Ser Tyr Asn Gln Gln 35 40 45 Gln Gln Ser Phe Asp Leu Ser Asn Gly Leu Ser Val Pro Ser Ala Ala 50 55 60 Gly Arg Leu Ser Asn Thr Tyr Gly Val Pro Ser Ala Gln Gly Ala Asn 65 70 75 80 Val Pro Ser Phe Asp Ser Ser Asp Ser Ile Ala Val Asp Ala Ala Gly 85 90 95 Arg Ser Gly Asn Ser Phe Ser Ser His Val Pro Ser Ser Thr Tyr Gly 100 105 110 Ala Pro Gly Asn Gly Phe Gly Gly Gly Ser Arg Ser Ser Gln Ser Gly 115 120 125 Ala Pro Ser Ser Val Tyr Gly Pro Pro Gln Ala Arg Asn Asn Asn Phe 130 135 140 Gly Asn Gly Ala Ala Pro Ser Ser Val Tyr Gly Pro Pro Gln Ala Arg 145 150 155 160 Asn Asn Asn Phe Gly Asn Gly Gly Ala Pro Ser Gln Val Tyr Gly Pro 165 170 175 Pro Lys Ala Arg Asn Asn Asn Phe Gly Asn Gly Ala Ala Pro Ser Ser 180 185 190 Val Tyr Gly Pro Pro Gln Ala Arg Asn Asn Asn Phe Gly Asn Gly Ala 195 200 205 Ala Pro Ser Ser Val Tyr Gly Pro Pro Gln Ala Arg Asn Asn Asn Phe 210 215 220 Ala Asn Ser Ala Ala Pro Ser Gln Val Tyr Gly Pro Pro Gln Ala Arg 225 230 235 240 Asn Asn Asn Phe Gly Asn Gly Ala Ala Pro Ser Ser Val Tyr Gly Pro 245 250 255 Pro Gln Ser Ser Ser Phe Ser Ser Pro Ser Gly Arg Ser Gly Gln Leu 260 265 270 Pro Ser Ala Thr Tyr Gly Ala Pro Phe Glu Arg Asn Gly Phe Gly Ser 275 280 285 Gln Gly Ser Ser Gly Phe Gln Gly Tyr Glu Pro Ser Lys Arg Ser Gln 290 295 300 Thr Thr Glu Asp Pro Phe Ala Glu Pro Ala Lys Tyr Glu Tyr Asp Tyr 305 310 315 320 Lys Val Gln Ala Ser Asp Glu Thr Gly Thr Glu Phe Gly His Lys Glu 325 330 335 Ser Arg Glu Asn Glu Ser Ala Arg Gly Ala Tyr His Val Leu Leu Pro 340 345 350 Asp Gly Arg Met Gln Ile Val Gln Tyr Glu Ala Asp Glu Thr Gly Tyr 355 360 365 Arg Pro Gln Ile Arg Tyr Glu Asp Thr Gly Tyr Pro Ser Ala Ala Ser 370 375 380 Ser Arg Ser Asn Asn Gly Phe Asn Gly Tyr Gln Tyr 385 390 395 <210> 20 <211> 573 <212> PRT <213> Anopheles gambiae <400> 20 Lys Arg Glu Ala Pro Leu Pro Pro Ser Gly Ser Tyr Leu Pro Pro Ser 1 5 10 15 Gly Gly Ala Gly Gly Tyr Pro Ala Ala Gln Thr Pro Ser Ser Ser Tyr 20 25 30 Gly Ala Pro Thr Gly Gly Ala Gly Ser Trp Gly Gly Asn Gly Gly Asn 35 40 45 Gly Gly Arg Gly His Ser Asn Gly Gly Gly Ser Ser Phe Gly Gly Ser 50 55 60 Ala Pro Ser Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Phe Gly Gly 65 70 75 80 Gln Ser Ser Gly Gly Phe Gly Gly His Ser Ser Gly Gly Phe Gly Gly 85 90 95 His Ser Ser Gly Gly His Gly Gly Asn Gly Asn Gly Asn Gly Asn Gly 100 105 110 Tyr Ser Ser Gly Arg Pro Ser Ser Gln Tyr Gly Pro Pro Gln Gln Gln 115 120 125 Gln Gln Gln Gln Ser Phe Arg Pro Pro Ser Thr Ser Tyr Gly Val Pro 130 135 140 Ala Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ala Gln Gln His Ser Asn 145 150 155 160 Gly Gly Asn Gly Gly Tyr Ser Ser Gly Arg Pro Ser Thr Gln Tyr Gly 165 170 175 Ala Pro Ala Gln Ser Asn Gly Asn Gly Phe Gly Asn Gly Arg Pro Ser 180 185 190 Ser Ser Tyr Gly Ala Pro Ala Arg Pro Ser Thr Gln Tyr Gly Ala Pro 195 200 205 Ser Ala Gly Asn Gly Asn Gly Tyr Ala Gly Asn Gly Asn Gly Arg Ser 210 215 220 Tyr Ser Asn Gly Asn Gly Asn Gly His Gly Asn Gly His Ser Asn Gly 225 230 235 240 Asn Gly Asn Asn Gly Tyr Ser Arg Gly Pro Ala Arg Gln Pro Ser Gln 245 250 255 Gln Tyr Gly Pro Pro Ala Gln Ala Pro Ser Ser Gln Tyr Gly Ala Pro 260 265 270 Ala Gln Thr Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gln Thr Pro Ser 275 280 285 Cheese Gln Tyr Gly Ala Pro Ala Gln Thr Pro Cheese Cheese Gln Tyr Gly Ala 290 295 300 Pro Ala Gln Thr Pro Ser Ser Gln Tyr Gly Ala Pro Ala Pro Ser Arg 305 310 315 320 Pro Ser Gln Gln Tyr Gly Ala Pro Ala Pro Ser Arg Pro Ser Gln Gln 325 330 335 Tyr Gly Ala Pro Ala Gln Thr Pro Ser Ser Gln Tyr Gly Ala Pro Ala 340 345 350 Gln Thr Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gln Thr Pro Ser Ser 355 360 365 Gln Tyr Gly Ala Pro Ala Gln Thr Pro Cheese Cheese Gln Tyr Gly Ala Pro 370 375 380 Ala Gln Gln Pro Ser Ser Gln Tyr Gly Ala Pro Ala Pro Ser Arg Pro 385 390 395 400 Cheese Gln Gln Tyr Gly Ala Pro Ala Gln Gln Pro Cheese Ala Gln Tyr Gly 405 410 415 Ala Pro Ala Gln Thr Pro Ser Ser Gln Tyr Gly Ala Pro Ala Pro Ser 420 425 430 Arg Pro Ser Gln Gln Tyr Gly Ala Pro Ala Gln Ala Pro Ser Ser Gln 435 440 445 Tyr Gly Ala Pro Ala Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gln Gln 450 455 460 Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gln Thr Pro Ser Ser Gln Tyr 465 470 475 480 Gly Ala Pro Ser Phe Gly Pro Thr Gly Gly Ala Ser Phe Ser Ser Gly 485 490 495 Asn Gly Asn Val Gly Gly Ser Tyr Gln Val Ser Ser Thr Gly Asn Gly 500 505 510 Phe Ser Gln Ala Ser Phe Ser Ala Ser Ser Phe Ser Pro Asn Gly Arg 515 520 525 Thr Ser Leu Ser Ala Gly Gly Phe Ser Ser Gly Ala Pro Ser Ala Gln 530 535 540 Cheese Ala Gly Gly Tyr Cheese Cheese Gly Gly Pro Cheese Gln Val Pro Ala Thr 545 550 555 560 Leu Pro Gln Ser Tyr Ser Ser Asn Gly Gly Tyr Asn Tyr 565 570 <210> 21 <211> 513 <212> PRT <213> Glossina morsitans <400> 21 Arg Pro Glu Pro Pro Val Asn Thr Tyr Leu Pro Pro Ser Ala Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Ser Pro Leu Ala Pro Ser Asp Thr Tyr Gly Ala 20 25 30 Pro Gly Val Asn Gly Gly Gly Gly Gly Gly Gly Gly Pro Ser Ser Thr 35 40 45 Tyr Gly Ala Pro Gly Ser Gly Gly Gly Asn Gly Asn Gly Gly Gly Gly 50 55 60 Phe Gly Lys Pro Ser Ser Thr Tyr Gly Ala Pro Gly Leu Gly Gly Gly 65 70 75 80 Gly Asn Gly Gly Gly Arg Pro Ser Glu Thr Tyr Gly Ala Pro Ser Gly 85 90 95 Gly Gly Gly Asn Gly Phe Gly Lys Pro Ser Ser Thr Tyr Gly Ala Pro 100 105 110 Asn Gly Gly Gly Gly Asn Gly Gly Pro Gly Arg Pro Ser Ser Thr Tyr 115 120 125 Gly Ala Pro Gly Ser Gly Gly Gly Asn Gly Gly Ser Gly Arg Pro Ser 130 135 140 Ser Thr Tyr Gly Ala Pro Gly Leu Gly Gly Gly Asn Gly Gly Ser Gly 145 150 155 160 Arg Pro Ser Ser Met Tyr Gly Ala Pro Gly Leu Gly Gly Gly Asn Gly 165 170 175 Gly Ser Gly Arg Pro Ser Ser Thr Tyr Gly Ala Pro Gly Ser Gly Gly 180 185 190 Gly Asn Gly Gly Ser Gly Arg Pro Ser Ser Thr Tyr Gly Ala Pro Gly 195 200 205 Ser Gly Gly Gly Asn Gly Gly Ser Gly Arg Pro Ser Ser Thr Tyr Gly 210 215 220 Ala Pro Gly Asn Gly Asn Gly Gly Asn Gly Phe Gly Arg Pro Ser Ser 225 230 235 240 Thr Tyr Gly Ala Pro Gly Ser Gly Gly Ser Asn Gly Asn Gly Lys Pro 245 250 255 Ser Ser Thr Tyr Gly Ala Pro Gly Ser Gly Gly Gly Gly Gly Arg Pro 260 265 270 Ser Asp Ser Tyr Gly Pro Pro Ala Ser Gly Asn Gly Gly Arg Asn Gly 275 280 285 Asn Gly Asn Gly Gln Ser Gln Glu Tyr Leu Pro Pro Gly Gln Ser Gly 290 295 300 Ser Gly Gly Gly Gly Gly Tyr Gly Gly Gly Ser Gly Ser Gly Gly Ser 305 310 315 320 Gly Gly Gly Gly Gly Gly Gly Tyr Gly Gly Asp Gln Asp Asn Asn Val 325 330 335 Val Glu Tyr Glu Ala Asp Gln Glu Gly Tyr Arg Pro Gln Ile Arg Tyr 340 345 350 Glu Gly Asp Gly Ser Gln Gly Gly Phe Gly Gly Asp Gly Asp Gly Tyr 355 360 365 Ser Tyr Glu Gln Asn Gly Val Gly Gly Asp Gly Gly Gly Ala Gly Gly 370 375 380 Ala Gly Gly Tyr Ser Asn Gly Gln Asn Leu Gly Ala Asn Gly Tyr Ser 385 390 395 400 Ser Gly Arg Pro Asn Gly Gly Asn Gly Gly Gly Arg Arg Gly Gly Gly 405 410 415 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gln Asn Leu Gly Ser Asn Gly 420 425 430 Tyr Ser Ser Gly Ala Pro Asn Gly Phe Gly Gly Gly Asn Gly Gln Gly 435 440 445 Tyr Ser Gly Gly Arg Ser Asn Gly Asn Gly Gly Gly Gly Gly Gly Arg 450 455 460 Asn Gly Gly Arg Tyr Arg Asn Gly Gly Gly Gly Gly Gly Gly Arg Asn 465 470 475 480 Gly Gly Gly Ser Asn Gly Tyr Asn Tyr Asp Gln Pro Gly Ser Asn Gly 485 490 495 Phe Gly Arg Gly Gly Gly Asn Gly Glu Asn Asp Gly Ser Gly Tyr His 500 505 510 Tyr <210> 22 <211> 506 <212> PRT <213> Atta cephalotes <400> 22 Arg Ser Glu Pro Pro Val Asn Ser Tyr Leu His Pro Gly Ser Asp Thr 1 5 10 15 Ser Gly Thr Asn Gly Gly Arg Thr Asp Leu Ser Thr Gln Tyr Gly Ala 20 25 30 Pro Asp Phe Asn Asn Arg Gly Asn Gly Asn Ser Gly Ala Thr Ser Phe 35 40 45 Gly Gly Ser Gly Ala Gly Asn Gly Pro Ser Lys Leu Tyr Asp Val Pro 50 55 60 Ile Arg Gly Asn Thr Gly Gly Asn Gly Leu Gly Gln Phe Arg Gly Asn 65 70 75 80 Gly Phe Glu Ser Gly Gln Pro Ser Ser Ser Tyr Gly Ala Pro Lys Gly 85 90 95 Gly Phe Gly Glu Asn Arg Gly Asn Arg Gly Arg Pro Ser Thr Ser Tyr 100 105 110 Gly Val Pro Asp Ser Asn Arg Asn Asn Arg Gly Gly Phe Gly Asn Gly 115 120 125 Gly Ser Glu Ala Arg Pro Ser Thr Ser Tyr Gly Val Pro Gly Ala Asn 130 135 140 Gly Asn Gln Gly Gly Phe Gly Ser Gly Ser Ile Gly Gly Arg Pro Ser 145 150 155 160 Thr Ser Tyr Gly Val Pro Gly Ala Asn Gly Asn Asn Gly Asp Ser Phe 165 170 175 Arg Asn Gly Asp Ile Gly Gly Arg Pro Ser Thr Asn Tyr Gly Ala Pro 180 185 190 Gly Ala Asn Gly Asn His Gly Gly Gly Asn Gly Gly Asn Gly Arg Pro 195 200 205 Ser Asn Asn Tyr Gly Val Pro Gly Ala Asn Gly Asn Thr Asn Gly Lys 210 215 220 Gly Arg Leu Asn Gly Asn Ser Gly Gly Gly Pro Ser Asn Asn Tyr Gly 225 230 235 240 Ser Pro Asn Gly Phe Gly Lys Gly Leu Ser Thr Ser Tyr Gly Ser Pro 245 250 255 Asn Arg Gly Gly Asn Asp Asn His Tyr Pro Ser Arg Gly Ser Phe Ile 260 265 270 Asn Gly Gly Ile Asn Gly Tyr Ser Ser Gly Ser Pro Asn Gly Asn Ala 275 280 285 Gly Asn Phe Gly His Gly Asp Glu Ser Phe Gly Arg Gly Gly Gly Glu 290 295 300 Gly Glu Asn Thr Gly Glu Gly Tyr Asn Ala Asn Ala Gln Glu Glu Ser 305 310 315 320 Thr Glu Pro Ala Lys Tyr Glu Phe Ser Tyr Lys Val Lys Asp Gln Gln 325 330 335 Thr Gly Ser Asp Tyr Ser His Thr Glu Thr Arg Asp Gly Asp His Ala 340 345 350 Gln Gly Glu Phe Asn Val Leu Leu Pro Asp Gly Arg Lys Gln Ile Val 355 360 365 Glu Tyr Glu Ala Asp Gln Asp Gly Phe Lys Pro Gln Ile Arg Tyr Glu 370 375 380 Gly Glu Ala Asn Ala Asp Gly Gly Tyr Gly Ser Gly Leu Asn Asp Asn 385 390 395 400 Asn Asp Gly Tyr Ser Ser Gly Arg Pro Asp Ser Glu Ser Gly Gly Phe 405 410 415 Ala Asn Ser Gly Phe Asn Gly Gly Ser Ser Asn Gly Gly Tyr Pro Asn 420 425 430 Gly Gly Pro Gly Glu Arg Lys Leu Gly Gly Phe Asn Asn Gly Gly Ser 435 440 445 Ser Gly Tyr Gln Ser Gly Arg Ser Ala Gly Gln Ser Phe Gly Arg Asp 450 455 460 Asn Ala Gly Asp Leu Asn Asn Asp Ile Gly Gly Tyr Phe Ser Asn Ser 465 470 475 480 Pro Asn Asn Ile Gly Asp Ser Asp Asn Ala Asn Val Gly Ser Asn Arg 485 490 495 Gln Asn Asp Gly Asn Ser Gly Tyr Gln Tyr 500 505 <210> 23 <211> 550 <212> PRT <213> Anopheles darlingi <400> 23 Lys Arg Glu Ala Pro Leu Pro Pro Ser Gly Ser Tyr Leu Pro Pro Ser 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Tyr Pro Ala Ala Gln Thr Pro Ser 20 25 30 Ser Ser Tyr Gly Ala Pro Ala Gly Gly Ala Gly Gly Trp Gly Gly Asn 35 40 45 Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly Arg Gly Gly Tyr Ser Asn 50 55 60 Gly Gly Gly His Ser Gly Ser Ala Pro Ser Gln Ser Tyr Gly Ala Pro 65 70 75 80 Ser Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Gln Ser Tyr Gly Ala 85 90 95 Pro Ala Ala Ala Pro Ser Gln Ser Tyr Gly Ala Pro Ser Phe Gly Gly 100 105 110 Asn Gly Gly Gly Ala Ser His Gly Ser Gly Gly Phe Thr Gly Gly His 115 120 125 Gly Gly Asn Gly Asn Gly Asn Gly Tyr Ser Ser Gly Arg Pro Ser Ser 130 135 140 Gln Tyr Gly Pro Pro Gln Gln Gln Gln Gln Pro Gln Gln Gln Ser Phe 145 150 155 160 Arg Pro Pro Ser Thr Ser Tyr Gly Val Pro Ala Ala Pro Ser Ser Ser 165 170 175 Tyr Gly Ala Pro Ser Ala Asn Gly Phe Ser Asn Gly Gly Arg Pro Ser 180 185 190 Ser Gln Tyr Gly Ala Pro Ala Pro Gln Ser Asn Gly Asn Glu Phe Gly 195 200 205 Ala Pro Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ser Arg Pro Ser Thr 210 215 220 Gln Tyr Gly Ala Pro Ser Asn Gly Asn Gly Asn Gly Tyr Ala Gly His 225 230 235 240 Gly Asn Gly Asn Gly His Gly Asn Gly Asn Gly His Ser Asn Gly Asn 245 250 255 Gly Asn Gly Tyr Asn Arg Gly Pro Ala Arg Gln Pro Ser Ser Gln Tyr 260 265 270 Gly Pro Pro Ser Gln Gly Pro Pro Ser Ser Gln Tyr Gly Pro Pro Ser 275 280 285 Gln Tyr Gly Pro Pro Ser Ser Gly Thr Ser Phe Ile Ala Tyr Gly Pro 290 295 300 Pro Ser Gln Gly Pro Pro Ser Ser Gln Tyr Gly Ala Pro Ala Pro Ser 305 310 315 320 Arg Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gln Thr Pro Ser Ser Gln 325 330 335 Tyr Gly Ala Pro Ala Gln Thr Pro Ser Ser Gln Tyr Gly Pro Pro Arg 340 345 350 Gln Ser Ser Pro Gln Phe Gly Ala Pro Ala Pro Arg Pro Pro Ser Ser 355 360 365 Gln Tyr Gly Ala Pro Ala Gln Ala Pro Cheese Cheese Gln Tyr Gly Ala Pro 370 375 380 Ala Gln Thr Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gln Ala Pro Ser 385 390 395 400 Cheese Gln Tyr Gly Ala Pro Ala Pro Cheese Arg Pro Cheese Cheese Gln Tyr Gly 405 410 415 Val Pro Ala Gln Ala Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gln Ala 420 425 430 Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gln Thr Pro Ser Ser Gln Tyr 435 440 445 Gly Ala Pro Ser Phe Gly Ser Thr Gly Gly Ser Ser Phe Gly Gly Asn 450 455 460 Gly Gly Val Gly Gly Ser Tyr Gln Thr Ala Ser Ser Gly Asn Gly Phe 465 470 475 480 Ser Gln Ala Ser Phe Ser Ala Ser Ser Phe Ser Ser Asn Gly Arg Ser 485 490 495 Ser Gln Ser Ala Gly Gly Tyr Ser Ser Gly Gly Pro Ser Gln Val Pro 500 505 510 Ala Thr Ile Pro Gln Gln Tyr Ser Ser Gly Gly Gly Ser Tyr Ser Ser 515 520 525 Gly Gly His Ser Gln Val Pro Ala Thr Leu Pro Gln Gln Tyr Ser Ser 530 535 540 Asn Gly Gly Tyr Asn Tyr 545 550 <210> 24 <211> 585 <212> PRT <213> Acromyrmex echinatior <400> 24 Arg Ser Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Gly Pro Gly Thr 1 5 10 15 Ser Gly Ala Asn Gly Gly Gln Thr Asp Leu Ser Ile Gln Tyr Arg Ala 20 25 30 Ser Asp Phe Asn Asn Arg Gly Asn Val Asn Gly Asn Ser Gly Ala Thr 35 40 45 Ser Phe Gly Gly Pro Gly Ala Ser Asn Gly Pro Ser Lys Leu Tyr Asp 50 55 60 Val Pro Ile Gly Gly Asn Ala Gly Gly Asn Gly Leu Gly Gln Phe Arg 65 70 75 80 Gly Asn Gly Phe Glu Gly Gly Gln Pro Ser Ser Ser Tyr Gly Ala Pro 85 90 95 Asn Gly Gly Phe Gly Glu Asn Arg Gly Asn Gly Gly Lys Pro Ser Thr 100 105 110 Ser Tyr Gly Val Pro Asp Ser Asn Gly Asn Asn Arg Gly Gly Phe Gly 115 120 125 Asn Gly Gly Ser Glu Gly Arg Pro Ser Thr Ser Tyr Gly Leu Pro Asp 130 135 140 Ala Ser Arg Asn Asn Gly Asn Gly Phe Gly Asn Val Gly Asn Glu Asp 145 150 155 160 Lys Pro Ser Thr Asn Tyr Gly Ile Pro Ala Asn Gly Asn Lys Val Ser 165 170 175 Gly Phe Gly Asn Val Gly Ser Glu Gly Arg Pro Ser Thr Ser Tyr Gly 180 185 190 Val Pro Gly Ala Asn Gly Asn Gln Gly Phe Gly Ser Gly Gly Ile Gly 195 200 205 Gly Arg Pro Ser Thr Ser Tyr Gly Val Pro Gly Val Asn Gly Asn Asn 210 215 220 Gly Gly Gly Phe Glu Asn Val Gly Arg Pro Ser Thr Ser Tyr Gly Thr 225 230 235 240 Pro Asp Ala Arg Gly Asn Asn Gly Gly Ser Phe Arg Asn Gly Asp Ile 245 250 255 Gly Gly Arg Pro Ser Thr Asn Tyr Gly Ile Pro Gly Ala Asn Gly Asn 260 265 270 His Gly Gly Gly Asn Gly Gly Asn Gly Arg Pro Ser Ser Asn Tyr Gly 275 280 285 Val Pro Gly Gly Asn Gly Asn Thr Asn Gly Lys Gly Arg Phe Asn Gly 290 295 300 Asn Ser Gly Gly Arg Pro Ser Asn Ser Tyr Gly Ser Pro Asn Gly Phe 305 310 315 320 Gly Lys Gly Leu Ser Thr Ser Tyr Ser Pro Ser Asn Arg Asp Gly Asn 325 330 335 Gly Asn His Tyr Pro Ser Gly Asp Ser Asn Arg Gly Ser Phe Val Asn 340 345 350 Gly Gly Ile Asn Gly Tyr Pro Ser Gly Ser Pro Asn Gly Asn Ala Gly 355 360 365 Asn Phe Arg His Gly Asp Glu Ser Phe Gly Arg Gly Gly Glu Gly Gly 370 375 380 Gly Arg Ser Thr Gly Glu Gly Tyr Asn Ala Asn Ala Gln Glu Glu Ser 385 390 395 400 Thr Glu Pro Ala Lys Tyr Glu Phe Ser Tyr Lys Val Lys Asp Gln Gln 405 410 415 Thr Gly Ser Asp Tyr Ser His Thr Glu Thr Arg Asp Gly Asp His Ala 420 425 430 Gln Gly Glu Phe Asn Val Leu Leu Pro Asp Gly Arg Lys Gln Ile Val 435 440 445 Glu Tyr Glu Ala Asp Gln Asp Gly Phe Lys Pro Gln Ile Arg Tyr Glu 450 455 460 Gly Glu Ala Asn Ala Asp Gly Glu Tyr Asp Ser Gly Gly Leu Asn Asp 465 470 475 480 Asn Asn Asp Gly Tyr Ser Ser Gly Arg Pro Gly Ser Glu Ser Gly Gly 485 490 495 Phe Ala Asn Asn Ser Gly Phe Asn Gly Gly Ser Ser Asn Gly Gly Tyr 500 505 510 Pro Ser Gly Gly Ser Gly Glu Gly Lys Leu Gly Phe Asn Ser Gly Gly 515 520 525 Asn Ser Gly Tyr Gln Ser Gly Arg Pro Ala Gly Gln Ser Phe Gly Arg 530 535 540 Asp Asn Ala Gly Asp Leu Ser Asn Asp Ile Gly Gly Phe Ser Asn Ser 545 550 555 560 Pro Asn Asn Ile Gly Gly Asp Asn Ala Asn Val Gly Ser Asn Arg Gln 565 570 575 Asn Gly Gly Asn Ser Gly Tyr Gln Tyr 580 585 <210> 25 <211> 748 <212> PRT <213> Acyrthosiphon pisum <400> 25 Glu Ser Pro Tyr Gly Gly Gly Ser Ser Asn Ser Asn Gly Asn Gly Arg 1 5 10 15 Asn Gly Gly Tyr Gly Gly Lys Gly Gln Tyr Gly Gly Gly Asn Gly Gly 20 25 30 Gly Val Gly Ser Ser Ser Ala Ser Pro Phe Phe Ser Gly Ala Asn Gln 35 40 45 Tyr Gly Ser Gln Ser Gly Leu Ser Gly Ala Ala Asn Asn Arg Tyr Pro 50 55 60 Ser Phe Gly Ser Lys Phe Gly Gly Asn Lys Gly Ser Tyr Gly Gly Ser 65 70 75 80 Ser Ser Arg Asn Asn Gly Arg Tyr Gly Ser Gly Ser Ala Ser Gly Tyr 85 90 95 Gly Ser Gly Ser Ser Gly Gly Leu Gly Ser Thr Gly Arg Ser Thr Gly 100 105 110 Gly Tyr Gly Gly Gly Ser Ser Gly Ser Tyr Gly Ser Gly Ser Ser Gly 115 120 125 Ser Leu Gly Ser Ser Thr Gly Ser Asn Gly Ile Tyr Gly Ala Gly Ser 130 135 140 Ser Gly Gly Phe Gly Ser Gly Ser Ser Gly Ser Tyr Gly Gly Gly Ser 145 150 155 160 Ser Gly Gly Phe Gly Ser Gly Ser Ser Gly Ser Tyr Gly Gly Gly Ser 165 170 175 Ser Gly Gly Phe Gly Ser Gly Ser Ser Gly Ser Tyr Gly Gly Gly Ser 180 185 190 Ser Gly Gly Phe Gly Ser Gly Ser Ser Gly Ser Tyr Gly Gly Gly Ser 195 200 205 Ser Gly Gly Phe Gly Ser Gly Ser Ser Gly Asn Tyr Gly Ser Gly Ser 210 215 220 Ser Gly Ser Tyr Gly Ser Gly Gly Gly Gly Leu Gly Gly Ala Ser Ser 225 230 235 240 Gly Asn Asn Asp Gly Tyr Gly Ala Gly Gly Ser Gly Ser Tyr Asp Gln 245 250 255 Leu Gly Gly Ala Asn Gly Asn Gly Leu Gly Gly Ser Gly Asn Asp Pro 260 265 270 Leu Ser Glu Pro Ala Asn Tyr Glu Phe Ser Tyr Glu Val Asn Ala Pro 275 280 285 Glu Ser Gly Ala Ile Phe Gly His Lys Glu Ser Arg Gln Gly Glu Glu 290 295 300 Ala Thr Gly Val Tyr His Val Leu Leu Pro Asp Gly Arg Thr Gln Ile 305 310 315 320 Val Glu Tyr Glu Ala Asp Glu Asp Gly Tyr Lys Pro Lys Ile Thr Tyr 325 330 335 Thr Asp Pro Val Gly Gly Tyr Ala Gly Asp Arg Gln Ser Gly Asn Ser 340 345 350 Tyr Gly Gly Asn Gly Gly Phe Gly Gly Ser Gly Ser Leu Gly Gly Ser 355 360 365 Gly Gly Asn Leu Gly Gly Leu Tyr Asn Gly Gly Gly Ser Ser Asn Asn 370 375 380 Gly Ala Gly Tyr Gly Gly Ser Ser Ser Ser Leu Gly Ser Arg Tyr Gly 385 390 395 400 Gly Ser Gly Gly Ser Ser Gly Ser Gly Val Gly Gly Gly Tyr Gly Gly 405 410 415 Ser Gly Ser Ser Ser Gly Gly Ile Gly Ser Ser Tyr Gly Gly Ser Gly 420 425 430 Ser Leu Ser Gly Gly Leu Gly Gly Gly Tyr Gly Gly Ser Gly Ser Ser 435 440 445 Ser Gly Gly Leu Gly Gly Gly Tyr Gly Gly Ser Gly Gly Ser Ser Gly 450 455 460 Gly Gly Phe Gly Gly Leu Gly Gly Ser Gly Gly Ser Ser Gly Ser Gly 465 470 475 480 Tyr Gly Gly Ser Gly Ser Ser Ser Gly Gly Leu Gly Asn Ser Tyr Gly 485 490 495 Gly Ser Gly Ser Ser Asn Gly Gly Leu Gly Gly Gly Tyr Ser Gly Ser 500 505 510 Gly Gly Ser Ser Gly Gly Leu Gly Gly Gly Tyr Gly Ala Ser Ser Gly 515 520 525 Ser Ser Gly Ser Gly Leu Gly Gly Gly Tyr Gly Gly Ser Gly Ser Ser 530 535 540 Ser Gly Gly Leu Gly Ser Gly Tyr Gly Gly Leu Gly Ser Ser Ser Gly 545 550 555 560 Gly Leu Gly Gly Gly Tyr Gly Gly Ser Gly Ser Ser Ser Gly Gly Leu 565 570 575 Gly Gly Gly Tyr Gly Gly Ser Gly Ser Ser Asn Gly Gly Ile Gly Gly 580 585 590 Gly Tyr Gly Gly Ser Ser Gly Ser Ser Gly Gly Leu Gly Gly Gly Tyr 595 600 605 Gly Gly Ser Gly Ser Ser Ser Gly Gly Leu Gly Gly Gly Tyr Gly Gly 610 615 620 Ser Gly Gly Ser Asn Ser Gly Leu Gly Ser Ser Tyr Gly Gly Ser Gly 625 630 635 640 Ser Thr Asn Gly Gly Leu Gly Gly Gly Tyr Gly Gly Leu Gly Ser Ser 645 650 655 Ser Gly Gly Leu Gly Gly Gly Tyr Gly Gly Ser Gly Gly Ser Asn Gly 660 665 670 Gly Ile Gly Gly Gly Tyr Gly Gly Ser Ser Gly Ser Gly Gly Ser Gln 675 680 685 Gly Ser Ala Tyr Gly Gly Ser Gly Ser Ser Ser Gly Ser Gln Gly Gly 690 695 700 Gly Tyr Gly Gly Ser Gly Ser Ser Ser Gly Gly Leu Gly Gly Gly Tyr 705 710 715 720 Gly Ser Ser Ser Gly Ser Ser Ser Gly Leu Gly Gly Ser Tyr Gly Ser 725 730 735 Asn Arg Asn Gly Leu Gly Ser Gly Ser Ser Tyr Ser 740 745 <210> 26 <211> 616 <212> PRT <213> Drosophila virilis <400> 26 Arg Pro Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Ser Pro Gly Asp 1 5 10 15 Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Gln Gly Gln Gly Gly Phe 20 25 30 Gly Gly Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ala Gly Asn Gly 35 40 45 Asn Gly Asn Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly 50 55 60 Gln Gly Gln Gly Gly Phe Gly Gly Lys Pro Ser Asp Ser Tyr Gly Ala 65 70 75 80 Pro Gly Ala Gly Asn Gly Asn Gly Asn Gly Arg Pro Ser Ser Ser Tyr 85 90 95 Gly Ala Pro Gly Gln Gly Gln Gly Gln Gly Gly Phe Gly Gly Arg Pro 100 105 110 Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Gly Phe Gly Gly 115 120 125 Lys Pro Ser Asp Thr Tyr Gly Ala Pro Gly Ala Gly Asn Gly Asn Gly 130 135 140 Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Gly Ile 145 150 155 160 Gly Gly Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ala Gly Asn Gly 165 170 175 Asn Gly Asn Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly 180 185 190 Gln Gly Gly Phe Gly Gly Lys Pro Ser Asp Thr Tyr Gly Ala Pro Gly 195 200 205 Ala Gly Asn Gly Asn Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly 210 215 220 Gln Gly Gln Gly Gly Phe Gly Gly Lys Pro Ser Asp Thr Tyr Gly Ala 225 230 235 240 Pro Gly Ala Gly Asn Gly Asn Gly Asn Gly Arg Pro Ser Ser Ser Tyr 245 250 255 Gly Ala Pro Gly Gln Gly Gln Gly Gly Phe Gly Gly Lys Pro Ser Asp 260 265 270 Thr Tyr Gly Ala Pro Gly Ala Gly Asn Gly Asn Gly Arg Pro Ser Ser 275 280 285 Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Gln Gly Gly Phe Gly Gly 290 295 300 Lys Pro Ser Asp Ser Tyr Gly Pro Pro Ala Ser Gly Ala Gly Ala Gly 305 310 315 320 Gly Ala Gly Gly Pro Gly Ala Gly Gly Gly Gly Asp Tyr Asp Asn Asp 325 330 335 Glu Pro Ala Lys Tyr Glu Phe Asn Tyr Gln Val Glu Asp Ala Pro Ser 340 345 350 Gly Leu Ser Phe Gly His Ser Glu Met Arg Asp Gly Asp Phe Thr Thr 355 360 365 Gly Gln Tyr Asn Val Leu Leu Pro Asp Gly Arg Lys Gln Ile Val Glu 370 375 380 Tyr Glu Ala Asp Gln Gln Gly Tyr Arg Pro Gln Val Arg Tyr Glu Gly 385 390 395 400 Asp Ala Asn Gly Asn Gly Gly Pro Gly Gly Ala Gly Gly Pro Gly Gly 405 410 415 Gln Asp Leu Gly Gln Asn Gly Tyr Ser Ser Gly Arg Pro Gly Gly Gln 420 425 430 Asp Leu Gly Gln Gly Gly Tyr Ser Asn Gly Arg Pro Gly Gly Gln Asp 435 440 445 Leu Gly Gln Asn Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu 450 455 460 Gly Gln Asn Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly 465 470 475 480 Gln Asn Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Gln 485 490 495 Asn Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Gln Asn 500 505 510 Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Gln Asn Gly 515 520 525 Tyr Ser Gly Gly Arg Pro Gly Gly Asn Gly Gly Ser Asp Gly Gly Arg 530 535 540 Val Ile Ile Gly Gly Arg Val Ile Gly Gln Asp Gly Gly Asp Gly Gln 545 550 555 560 Gly Tyr Ser Ser Gly Arg Pro Asn Gly Gln Asp Gly Gly Phe Gly Gln 565 570 575 Asp Asn Thr Asp Gly Arg Gly Tyr Ser Ser Gly Lys Pro Gly Gln Gly 580 585 590 Arg Asn Gly Asn Gly Asn Ser Phe Gly Pro Gly Gly Gln Asn Gly Asp 595 600 605 Asn Asp Gly Ser Gly Tyr Arg Tyr 610 615 <210> 27 <211> 557 <212> PRT <213> Drosophila erecta <400> 27 Arg Pro Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Ser Asp Ser Tyr 1 5 10 15 Gly Ala Pro Gly Gln Ser Gly Pro Gly Gly Arg Pro Ser Asp Ser Tyr 20 25 30 Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp Ser Tyr Gly 35 40 45 Ala Pro Gly Leu Gly Gln Gly Gln Gly Gln Gly Gln Gly Gln Gly Gly 50 55 60 Phe Gly Gly Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ala Gly Asn 65 70 75 80 Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ala Gly 85 90 95 Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Ser 100 105 110 Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly 115 120 125 Asn Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gln 130 135 140 Gly Gln Gly Asn Gly Asn Ser Gly Arg Pro Ser Ser Ser Tyr Gly Ala 145 150 155 160 Pro Gly Ala Gly Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro 165 170 175 Gly Gly Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly 180 185 190 Ala Gly Asn Gly Gly Asn Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala 195 200 205 Pro Gly Gly Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Ser Gly 210 215 220 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Gly 225 230 235 240 Phe Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln Asn Gln 245 250 255 Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly Ser Gly Ser Gly 260 265 270 Asn Gly Asn Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser 275 280 285 Gly Pro Gly Gly Arg Pro Ser Asp Ser Tyr Gly Pro Pro Ala Ser Gly 290 295 300 Ser Gly Ala Gly Gly Ala Gly Gly Ser Gly Pro Gly Gly Ala Asp Tyr 305 310 315 320 Asp Asn Asp Ile Val Glu Tyr Glu Ala Asp Gln Gln Gly Tyr Arg Pro 325 330 335 Gln Ile Arg Tyr Glu Gly Asp Ala Asn Asp Gly Ser Gly Pro Ser Gly 340 345 350 Pro Gly Gly Gln Asn Leu Gly Ala Asp Gly Tyr Ser Ser Gly Arg Pro 355 360 365 Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly Tyr Ser Gly Gly Arg Pro 370 375 380 Gly Gly Gln Asp Leu Gly Pro Ser Gly Tyr Ser Gly Gly Arg Pro Gly 385 390 395 400 Gly Gln Asp Leu Gly Ala Gly Gly Tyr Ser Asn Gly Arg Pro Gly Gly 405 410 415 Gln Asp Leu Gly Pro Ser Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln 420 425 430 Asp Leu Gly Pro Ser Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp 435 440 445 Leu Gly Ala Gly Gly Tyr Ser Asn Gly Arg Pro Gly Gly Asn Gly Asn 450 455 460 Gly Asn Gly Gly Ala Asp Gly Gly Arg Val Ile Ile Gly Gly Arg Val 465 470 475 480 Ile Gly Gly Gln Asp Gly Gly Asp Gln Gly Tyr Ser Gly Gly Arg Pro 485 490 495 Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Ser Gly Arg Pro Gly 500 505 510 Gly Arg Pro Gly Ala Asn Gly Gln Asp Asn Gln Asp Gly Gln Gly Tyr 515 520 525 Ser Ser Gly Arg Ser Gly Lys Gly Gly Arg Asn Ser Phe Gly Pro Gly 530 535 540 Gly Gln Asn Gly Asp Asn Asp Gly Ser Gly Tyr Arg Tyr 545 550 555 <210> 28 <211> 796 <212> PRT <213> Lutzomyia longipalpis <400> 28 Arg Pro Glu Pro Pro Ala Asn Thr Tyr Leu Pro Pro Ser Ser Ser Tyr 1 5 10 15 Ala Ala Pro Gly Gln Gln Gly Gly Ser Gly Phe Gly Gly Gly Gly Gly 20 25 30 Ser Gly Gly Ser Gly Gly Phe Gly Gln Pro Gly Ala Phe Gly Arg Pro 35 40 45 Ser Ser Ser Tyr Gly Pro Pro Ser Gln Gly Gly Ala Gly Gly Gly Phe 50 55 60 Gly Ser Asp Ser Gln Phe Gly Gly Gly Phe Gly Gly Gly Ala Gly Gly 65 70 75 80 Phe Gly Ser Gly Gly Ser Gly Ala Pro Gly Ala Ser Gln Arg Pro Ser 85 90 95 Ser Ser Tyr Gly Pro Pro Gly Gln Thr Gly Gly Gly Gly Phe Gly Ala 100 105 110 Gln Gly Ala Pro Gly Ser Ser Phe Gly Pro Gly Gly Gly Phe Gly Gly 115 120 125 Gly Ser Pro Gly Gln Ala Gly Ser Pro Gly Phe Gln Arg Pro Ser Ser 130 135 140 Ser Tyr Gly Pro Pro Gly Gln Ser Pro Gly Gly Gly Phe Ser Gln Gln 145 150 155 160 Gly Gly Ala Pro Gly Ala Ser Gln Arg Pro Ser Ser Thr Tyr Gly Ala 165 170 175 Pro Gly Gln Gly Ala Gly Gly Phe Gly Gln Gly Gly Ser Gly Gly Phe 180 185 190 Gly Gly Thr Gly Gly Ser Val Ala Ile Gly Gly Arg Pro Ser Ser Ser 195 200 205 Tyr Gly Ala Pro Gly Gln Gly Ser Ser Gly Gly Phe Gly Gly Gly Ser 210 215 220 Gly Gly Phe Gly Ser Gln Ala Pro Ser Thr Ser Tyr Gly Ala Pro Gly 225 230 235 240 Gln Gly Ser Pro Gly Gly Gly Phe Gly Ser Gln Gly Gly Pro Gly Gly 245 250 255 Gln Pro Gly Ser Pro Gly Phe Gly Gly Ser Gln Arg Pro Ser Ser Ser 260 265 270 Tyr Gly Pro Pro Gly Gln Gly Gly Ala Pro Gly Gln Gly Gly Ser Pro 275 280 285 Gly Phe Gly Ala Ser Ser Arg Ser Gly Gly Ala Gly Gly Phe Gly Ala 290 295 300 Ser Gln Gln Pro Ser Ser Ser Tyr Gly Pro Pro Gly Gln Gly Ala Gly 305 310 315 320 Ser Gly Phe Gln Gly Thr Gly Gly Gly Phe Gly Gly Pro Gly Gln Arg 325 330 335 Pro Gly Phe Gly Gly Ser Gln Thr Pro Ala Thr Ser Tyr Gly Ala Pro 340 345 350 Gly Gln Ala Gly Gly Ala Ser Gly Gly Phe Gly Gly Ala Gly Ala Gln 355 360 365 Arg Pro Ser Ser Ser Tyr Gly Pro Pro Gly Gln Ala Ser Gly Phe Gly 370 375 380 Gly Gly Ser Ser Gly Gly Gly Phe Gly Gly Gly Ser Ser Gly Gly Phe 385 390 395 400 Gly Gly Asn Gln Gly Gly Phe Gly Gly Asn Gln Gly Gly Phe Gly Gly 405 410 415 Ser Gln Thr Pro Ser Ser Ser Tyr Gly Ala Pro Ser Phe Gly Ser Gly 420 425 430 Gly Ser Pro Gly Ala Ala Gly Gly Ala Gly Gly Phe Gly Gln Gly Gly 435 440 445 Val Gly Gly Ser Gly Gln Pro Gly Gly Phe Gly Gly Gly Asp Gln Gly 450 455 460 Tyr Pro Pro Arg Gly Gly Pro Gly Gly Phe Gly Pro Gly Ser Gly Gly 465 470 475 480 Ser Gly Ala Gly Gly Pro Ile Ala Gly Gly Ser Gly Ser Gly Tyr Pro 485 490 495 Gly Gly Ser Asp Ser Gly Ser Asn Glu Pro Ala Lys Tyr Asp Phe Ser 500 505 510 Tyr Gln Val Asp Asp Pro Ala Ser Gly Thr Ser Phe Gly His Ser Glu 515 520 525 Gln Arg Asp Gly Asp Tyr Thr Ser Gly Gln Tyr Asn Val Leu Leu Pro 530 535 540 Asp Gly Arg Lys Gln Ile Val Glu Tyr Glu Ala Asp Leu Gly Gly Tyr 545 550 555 560 Arg Pro Gln Ile Lys Tyr Glu Gly Gly Ser Ser Gly Gly Ala Gly Gly 565 570 575 Tyr Pro Ser Gly Gly Pro Gly Ser Gln Gly Gly Ala Gly Gly Tyr Pro 580 585 590 Ser Gly Gly Pro Gly Gly Pro Gly Ser Pro Gly Gly Ala Gly Gly Tyr 595 600 605 Gln Ser Gly Ala Ala Gly Gly Ala Gly Gly Tyr Pro Ser Gly Gly Pro 610 615 620 Gly Gly Pro Gly Ala Gly Gly Tyr Pro Ser Gly Gly Pro Gly Gly Pro 625 630 635 640 Gly Ser Gln Ala Gly Gly Phe Ser Gly Gly Phe Gly Gly Gly Ser Asp 645 650 655 Gly Ala Phe Gly Gly Ala Gly Gly Phe Ser Gln Gly Gly Ala Gly Gly 660 665 670 Gly Asp Ala Gly Tyr Pro Arg Gly Gly Pro Gly Gly Phe Gly Gly Ala 675 680 685 Gly Ser Pro Gly Phe Gly Gly Ser Gly Ser Pro Gly Phe Gly Gly Ser 690 695 700 Gly Ser Pro Gly Ala Gln Gly Ser Ser Gly Phe Gly Gly Thr Gly Gly 705 710 715 720 Gly Phe Gly Gly Gly Ala Asp Gly Tyr Pro Arg Gly Gly Pro Gly Ala 725 730 735 Gly Gln Ser Gly Phe Gln Asp Gly Arg Gly Ala Thr Gly Gly Ala Gly 740 745 750 Gln Pro Gly Gly Arg Gly Ser Phe Gly Arg Pro Gly Ser Ala Arg Gly 755 760 765 Gly Ser Ser Ser Asn Gly Tyr Ala Asn Gly Gly Ala Glu Gly Tyr Pro 770 775 780 Arg Asp Asn Pro Gln Asn Arg Gly Ser Gly Tyr Ser 785 790 795 <210> 29 <211> 1051 <212> PRT <213> Rhodnius prolixus <400> 29 Lys Arg Asp Asp Pro Leu Arg Arg Phe Leu Ala Pro Leu Val Gly Gly 1 5 10 15 Gly Asn Gly Ser Gly Gly Gly Gly Gly Gly Tyr Asn Tyr Asn Lys Pro 20 25 30 Ala Asn Gly Leu Ser Leu Pro Gly Gly Gly Gly Ala Leu Pro Pro Ala 35 40 45 Thr Ser Tyr Gly Val Pro Asp Arg Pro Ala Pro Val Pro Ser Ser Pro 50 55 60 Pro Ser Ser Ser Tyr Gly Ala Pro Gln Pro Ser Pro Asn Tyr Gly Ala 65 70 75 80 Pro Ser Ser Ser Tyr Gly Ala Pro Ser Gln Gln Pro Ser Arg Ser Tyr 85 90 95 Gly Ala Pro Ser Gln Gly Pro Ser Thr Ser Tyr Ser Gln Arg Pro Ser 100 105 110 Ser Ser Tyr Gly Ala Pro Ala Pro Gln Thr Pro Ser Ser Ser Tyr Gly 115 120 125 Ala Pro Ala Gln Gln Pro Ser Gly Ser Tyr Gly Ala Pro Ser Gly Gly 130 135 140 Gly Gly Ser Ser Gly Tyr Thr Gly Gly Ala Gln Arg Pro Ser Gly Ser 145 150 155 160 Tyr Gly Ala Pro Ser Gln Gly Gly Pro Ser Gly Asn Tyr Gly Pro Pro 165 170 175 Gln Gln Pro Cheese Cheese Asn Tyr Gly Ala Pro Cheese Gln Thr Pro Cheese 180 185 190 Ser Asn Tyr Gly Ala Pro Ala Gln Arg Pro Ser Thr Ser Tyr Gly Ala 195 200 205 Pro Ser Gln Pro Pro Ser Ser Ser Ser Tyr Gly Ser Pro Pro Gln Arg Ala 210 215 220 Ser Gly Tyr Pro Ser Ser Ser Ser Gly Pro Ser Asn Gly Tyr Ser Pro 225 230 235 240 Pro Ala Gln Arg Pro Ser Ser Ser Tyr Gly Pro Pro Ser Gln Gln Pro 245 250 255 Ala Ser Ser Tyr Gly Ala Pro Ser Gln Thr Pro Ser Ser Asn Tyr Gly 260 265 270 Pro Pro Ala Pro Ile Pro Ser Ser Asn Tyr Gly Ala Pro Ser Gln Pro 275 280 285 Pro Ser Lys Pro Ser Ala Pro Ser Ser Ser Tyr Gly Thr Pro Ser Gln 290 295 300 Thr Pro Ser Thr Ser Tyr Gly Ala Pro Ser Gln Ala Pro Ser Ser Ser 305 310 315 320 Tyr Gly Ala Pro Ser Arg Pro Ser Pro Pro Ser Ser Ser Tyr Gly Ala 325 330 335 Pro Ser Gln Gly Pro Ser Ser Ser Ser Tyr Gly Pro Pro Ser Arg Pro Ser 340 345 350 Gln Pro Cheese Cheese Pro Cheese Cheese Gly Tyr Gly Ala Pro Cheese Gln Gly Pro 355 360 365 Cheese Ser Ser Tyr Gly Ala Pro Ser Arg Pro Ser Ser Pro Ser Ser Ser 370 375 380 Tyr Gly Ala Pro Pro Ser Ser Ser Tyr Gly Ala Pro Ser Arg Pro Ser 385 390 395 400 Pro Pro Ser Ser Ser Ser Tyr Gly Ala Pro Ser Gln Gly Pro Ser Ser Ser 405 410 415 Tyr Gly Pro Pro Ser Arg Pro Ser Gln Pro Ser Ser Pro Ser Ser Gly 420 425 430 Tyr Gly Ala Pro Ser Gln Gly Pro Ser Ser Ser Tyr Gly Ala Pro Ser 435 440 445 Arg Pro Ser Ser Pro Ser Ser Ser Ser Tyr Gly Ala Pro Pro Ser Ser Ser 450 455 460 Tyr Gly Ala Pro Ser Arg Pro Ser Pro Pro Ser Ser Ser Tyr Gly Ala 465 470 475 480 Pro Ser Gln Gly Pro Ser Ser Ser Ser Tyr Gly Pro Pro Ser Arg Pro Ser 485 490 495 Gln Pro Ser Ser Pro Ser Ser Ser Tyr Gly Ala Pro Ser Gln Gly Pro 500 505 510 Cheese Cheese Cheese Tyr Gly Ala Pro Cheese Arg Pro Cheese Pro Pro Cheese Cheese Cheese 515 520 525 Tyr Gly Ala Pro Ser Gln Gly Pro Ser Ser Ser Tyr Gly Pro Pro Ser 530 535 540 Arg Pro Ser Gln Pro Ser Ser Thr Tyr Gly Val Pro Ser Gly Gly Arg 545 550 555 560 Pro Ser Thr Pro Ser Ser Ser Tyr Gly Ala Pro Pro Gln Ala Leu Ser 565 570 575 Ser Thr Tyr Gly Ala Pro Ser Gly Arg Pro Gly Ala Pro Ser Gln Lys 580 585 590 Pro Ser Ser Ser Ser Tyr Gly Ala Pro Ser Leu Gly Gly Asn Ala Ser Arg 595 600 605 Gly Pro Lys Ser Ser Pro Pro Ser Ser Ser Tyr Gly Ala Pro Ser Val 610 615 620 Gly Thr Ser Val Ser Ser Tyr Ala Pro Ser Gln Gly Gly Ala Gly Gly 625 630 635 640 Phe Gln Ser Ser Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ser Thr Gly 645 650 655 Pro Ser Ser Thr Tyr Gly Pro Pro Ser Gln Pro Pro Ser Ser Ser Tyr 660 665 670 Gly Val Pro Ser Gln Pro Pro Ser Ser Asn Tyr Gly Val Pro Ser Gln 675 680 685 Gly Val Ser Gly Ser Val Gly Ser Ser Ser Pro Ser Ser Ser Tyr Gly 690 695 700 Ala Pro Ser Gln Ile Pro Ser Ser Ser Tyr Gly Ala Pro Ser Gln Ser 705 710 715 720 Cheese Ile Gly Gly Phe Gly Cheese Cheese Arg Pro Cheese Cheese Cheese Tyr Gly Ala 725 730 735 Pro Pro Gln Ala Pro Ser Ser Ser Tyr Ser Ala Pro Leu Arg Ala Pro 740 745 750 Ser Thr Ser Tyr Gly Ala Pro Ser Gly Gly Ser Gly Ser Asn Phe Gly 755 760 765 Ser Lys Pro Ser Thr Asn Tyr Gly Ala Pro Ser Gln Pro Pro Ser Thr 770 775 780 Asn Tyr Gly Pro Pro Ser Gln Pro Pro Ser Ser Ser Tyr Gly Thr Pro 785 790 795 800 Ser Arg Ala Pro Ser Pro Thr Tyr Ser Thr Pro Gln Ser Ser Gly Thr 805 810 815 Ser Phe Gly Ser Arg Pro Ser Ser Ser Tyr Gly Val Pro Ser Gln Pro 820 825 830 Thr Thr Asn Tyr Gly Ala Pro Ser Gln Thr Pro Ser Ser Asn Tyr Gly 835 840 845 Ala Pro Pro Ala Ser Ser Ala Pro Ser Ser Thr Tyr Gly Arg Pro Ser 850 855 860 Gln Ser Pro Ser Ser Ser Tyr Gly Ala Pro Ser Pro Ser Ser Ser Ser 865 870 875 880 Ser Ser Tyr Glu Ser Pro Ser Gln Pro Pro Ser Ser Ser Tyr Gly Ala 885 890 895 Pro Ser Gln Gly Pro Ser Ser Ser Tyr Gly Ala Pro Ser Arg Pro Ser 900 905 910 Ser Thr Tyr Gly Ala Pro Ser Pro Ser Ser Pro Ser Thr Asn Tyr Gly 915 920 925 Ala Pro Ala Pro Ser Ser Asn Tyr Gly Thr Pro Ala Gln Asp Leu Thr 930 935 940 Gly Ser Tyr Ala Ala Pro Ser Gln Pro Pro Ser Ala Gly Tyr Gly Ala 945 950 955 960 Pro Ser Gly Gln Pro Ser Ser Gly Gly Lys Gln Asn Phe Gln Val Lys 965 970 975 Asn Pro Phe Ala Gly Gln Thr His Gln Val Tyr Pro Ala Val Ser Ser 980 985 990 Ile Ser Phe Gly Leu Pro Ser Gln Ser Phe Asn Thr Ala Ile Gln Gly 995 1000 1005 Gln Glu Pro Ser Gln Ser Tyr Gly Ala Pro Thr Ala Ser Ser Pro 1010 1015 1020 Ser Ser Ser Tyr Gly Ala Pro Thr Gly Thr Gly Ser Ser Gln Pro 1025 1030 1035 Gly Gln Ser Tyr Ala Ser Asn Gly Gly Tyr Ser Tyr Ser 1040 1045 1050 <210> 30 <211> 506 <212> PRT <213> Rhodnius prolixus <400> 30 Gln Pro Pro Phe Asn His Tyr Leu Pro Ala Ala Arg Gly Ser Gly Ser 1 5 10 15 Asn Ser Ala Gln Tyr Thr Ala Pro Ser Ser Lys Phe Gly Thr Ser Thr 20 25 30 Gly Gln Tyr Gly Gln Pro Pro Ser Glu Val Pro Arg Gly Leu Gln Gln 35 40 45 Gly Ser Tyr Ala Glu Asp Val His Ser Ser Arg Ser Val Asn Pro Ser 50 55 60 Ser Gln Asn Gly Ile Pro Ser Gly His Phe Ser Ser Leu Ser Ser Asn 65 70 75 80 Tyr Gly Ala Pro Ser Ser Asp Tyr Ser Arg Ser Phe Leu Arg Tyr Gly 85 90 95 Thr Leu Ser Asn Lys Tyr Gly Val Pro Asn Ser Ala Leu Gly Ser Leu 100 105 110 Ser Ser Arg Asn Asn Lys Thr Pro Ala Thr Gln Leu Ser Tyr Gln Pro 115 120 125 Ser Ser His Tyr Asp Ser Arg Ser Thr Ser Glu Asp Gln Phe Ile Ser 130 135 140 Ser Arg Val Ser Asp Ser Gln Tyr Gly Ala Ser Ser Val Arg Arg Phe 145 150 155 160 Leu Pro Ser Ser Gln Tyr Ser Thr Pro Ser Ser Gln Tyr Gly Thr Pro 165 170 175 Ser Ser Gln Tyr Gly Thr Pro Ser Ser Gln Tyr Gly Thr Pro Ser Ser 180 185 190 Gln Tyr Gly Thr Pro Ser Ser Gln Tyr Gly Thr Pro Ser Ser Gln Tyr 195 200 205 Gly Thr Pro Ser Ser Gln Tyr Gly Thr Pro Ser Ser Gln Tyr Gly Thr 210 215 220 Pro Ser Ser Gln Tyr Gly Thr Pro Ser Ser Gln Tyr Gly Thr Pro Ser 225 230 235 240 Ser Pro Pro Ser Gln Tyr Gly Gly Pro Tyr Ser Met Arg Thr Ser Ala 245 250 255 Pro Asn Ser Gln Tyr Gly Thr Pro Ser Ser Phe Arg Thr Ser Pro Ser 260 265 270 Ser Gln Phe Gly Ser Ser Ser Ala His Ser Ser Ser Leu Ser Lys Phe 275 280 285 Arg Ser Val Pro Ser Ser Pro Tyr Gly Thr Leu Ser Ala Ile Arg Ser 290 295 300 Thr His Ser Ser Gln Tyr Gly Thr Pro Ser Ser Phe Ser Asp Ser Thr 305 310 315 320 Ser Ser Ser His Asn Gly Leu Pro Ser His Tyr Pro Gly Ser Gly Phe 325 330 335 Ser Gly Ser Ser Val Asn Asp Gln Lys Ser Tyr Thr Gly Asn Val Phe 340 345 350 Gly Gln Ser His Ser Arg Val Ala Asn Gly Asp Gln His Ala Arg Ser 355 360 365 Tyr Thr Leu Ala Gly Gly Asn Glu Ile Ser Glu Pro Ala Lys Tyr Asp 370 375 380 Phe Asn Tyr Asp Val Ser Asp Gly Glu Gln Gly Val Glu Phe Gly Gln 385 390 395 400 Glu Glu Ser Arg Asp Gly Glu Glu Thr Asn Gly Ser Tyr His Val Leu 405 410 415 Leu Pro Asp Gly Arg Arg Gln Arg Val Gln Tyr Thr Ala Gly Gln Tyr 420 425 430 Gly Tyr Lys Pro Thr Ile Ser Tyr Glu Asn Thr Gly Thr Leu Thr Thr 435 440 445 Gly Arg Gln Gln Phe Ser Asn Gly Phe Tyr Asn Val Gln Gln Ser Gly 450 455 460 Ser Glu Ser Gln Glu His Leu Gly Arg Ser Thr Gly Gln Asn Ser Tyr 465 470 475 480 Gly Gly Ser Asn Gly Tyr Glu Ser Gly Val Gly Tyr Gln Ser Gly Val 485 490 495 Gly Arg Arg Ser Arg Pro Ala Gly Ser Tyr 500 505 <210> 31 <211> 551 <212> PRT <213> Solenopsis invicta <400> 31 Arg Ser Glu Pro Pro Ile Asn Ser Tyr Leu Pro Pro Arg Ala Gly Ser 1 5 10 15 Ser Gly Ala Asn Gly Gly Arg Thr Asp Leu Thr Thr Gln Tyr Gly Ala 20 25 30 Pro Asp Phe Asn Asn Gly Gly Gly Ala Thr Ser Phe Ser Gly Asn Gly 35 40 45 Ala Gly Asp Gly Pro Ser Lys Leu Tyr Asp Val Pro Val Arg Gly Asn 50 55 60 Ala Gly Gly Asn Gly Leu Gly Arg Gly Asn Gly Phe Gly Gly Gly Gln 65 70 75 80 Pro Ser Ser Ser Tyr Gly Ala Pro Asn Gly Gly Ser Asn Glu Asn Arg 85 90 95 Gly Asn Gly Gly Arg Pro Ser Thr Ser Tyr Gly Val Pro Gly Ala Asn 100 105 110 Gly Asn Asn Gly Gly Gly Phe Gly Asn Gly Gly Asp Lys Gly Arg Pro 115 120 125 Ser Thr Ser Tyr Gly Val Pro Asp Ala Ser Gly Ser Ser Gln Gly Ser 130 135 140 Phe Gly Asn Val Gly Asn Gly Gly Arg Pro Ser Thr Asn Tyr Gly Val 145 150 155 160 Pro Gly Ala Asn Gly Asn Gly Gly Gly Phe Gly Asn Ala Ala Asn Glu 165 170 175 Gly Lys Pro Ser Thr Ser Tyr Gly Val Pro Gly Ala Asn Gly Asn Ser 180 185 190 Gln Gly Gly Phe Gly Asn Gly Gly Arg Pro Ser Thr Gly Tyr Gly Val 195 200 205 Pro Gly Ala Asn Gly Asn Asn Gly Gly Gly Phe Gly Gly Arg Pro Ser 210 215 220 Thr Ser Tyr Gly Ala Pro Gly Ala Asn Gly Asn His Arg Gly Gly Asn 225 230 235 240 Gly Gly Asn Ala Ser Pro Ser Thr Asn Tyr Gly Val Pro Gly Gly Asn 245 250 255 Asn Gly Asn Thr Asn Gly Lys Gly Arg Phe Asn Gly Gly Asn Ser Gly 260 265 270 Gly Gly Pro Ser Asn Asn Tyr Gly Val Pro Asn Glu Asn Ala Phe Gly 275 280 285 Gly Gly Leu Ser Thr Ser Tyr Gly Pro Pro Ser Arg Gly Gly Asn Gly 290 295 300 Asn Ser Gly Tyr Pro Ser Gly Gly Ser Asn Gly Gly Ser Phe Val Asn 305 310 315 320 Asn Gly Ala Asn Gly Tyr Pro Ser Gly Gly Pro Asn Gly Asn Ala Gly 325 330 335 Asn Phe Gly Asp Gly Arg Gly Gly Lys Gly Gly Gly Ser Ser Gly Glu 340 345 350 Gly Tyr Asn Asp Asn Ala Gln Glu Gly Ser Thr Glu Pro Ala Lys Tyr 355 360 365 Glu Phe Ser Tyr Lys Val Lys Asp Gln Gln Thr Gly Ser Glu Tyr Ser 370 375 380 His Thr Glu Thr Arg Asp Gly Asp Arg Ala Gln Gly Glu Phe Asn Val 385 390 395 400 Leu Leu Pro Asp Gly Arg Lys Gln Ile Val Glu Tyr Glu Ala Asp Gln 405 410 415 Asp Gly Phe Lys Pro Gln Ile Arg Tyr Glu Gly Glu Ala Asn Ala Gly 420 425 430 Gly Gly Tyr Ser Ser Gly Gly Ser Asn Asp Asn Asn Asp Gly Tyr Ser 435 440 445 Ser Gly Arg Pro Gly Ser Glu Ala Gly Gly Phe Ala Asn Asn Ser Gly 450 455 460 Phe Asn Gly Ser Gly Thr Asn Gly Gly Arg Ser Ser Gly Gly Pro Gly 465 470 475 480 Asp Gly Asn Pro Gly Gly Phe Asn Ser Gly Gly Gly Gly Gly Tyr Gln 485 490 495 Ser Gly Arg Pro Ala Gly Gln Ser Phe Gly Arg Asp Asn Asp Gly Gly 500 505 510 Leu Ser Gly Asp Ile Gly Gly Tyr Phe Ala Asn Ser Pro Ser Asn Asn 515 520 525 Ile Gly Gly Ser Asp Ser Ala Asn Val Gly Ser Asn Arg Gln Asn Gly 530 535 540 Gly Asn Gly Gly Tyr Gln Tyr 545 550 <210> 32 <211> 382 <212> PRT <213> Culex quinquefasciatus <400> 32 Lys Arg Glu Ala Pro Leu Pro Gly Gly Ser Tyr Leu Pro Pro Ser Asn 1 5 10 15 Gly Gly Gly Ala Gly Gly Tyr Pro Ala Ala Gly Pro Pro Ser Gly Ser 20 25 30 Tyr Gly Pro Pro Ser Asn Gly Asn Gly Asn Gly Asn Gly Ala Gly Gly 35 40 45 Tyr Pro Ser Ala Pro Ser Gln Gln Tyr Gly Ala Pro Ala Gly Gly Ala 50 55 60 Pro Ser Gln Gln Tyr Gly Ala Pro Ser Asn Gly Asn Gly Gly Ala Gly 65 70 75 80 Gly Tyr Pro Ser Ala Pro Ser Gln Gln Tyr Gly Ala Pro Asn Gly Asn 85 90 95 Gly Asn Gly Gly Phe Gly Gly Arg Pro Gln Ala Pro Ser Gln Gln Tyr 100 105 110 Gly Ala Pro Ser Asn Gly Asn Gly Gly Ala Arg Pro Ser Gln Gln Tyr 115 120 125 Gly Ala Pro Asn Gly Gly Asn Gly Asn Gly Arg Pro Gln Thr Pro Ser 130 135 140 Ser Gln Tyr Gly Ala Pro Ser Gly Gly Ala Pro Ser Ser Gln Tyr Gly 145 150 155 160 Ala Pro Ser Gly Gly Ala Pro Ser Gln Gln Tyr Gly Ala Pro Asn Gly 165 170 175 Gly Asn Gly Asn Gly Arg Pro Gln Thr Pro Ser Ser Gln Tyr Gly Ala 180 185 190 Pro Ser Gly Gly Ala Pro Ser Gln Gln Tyr Gly Ala Pro Asn Gly Gly 195 200 205 Asn Gly Asn Gly Arg Pro Gln Thr Pro Ser Ser Gln Tyr Gly Ala Pro 210 215 220 Ser Gly Gly Ala Pro Ser Ser Gln Tyr Gly Ala Pro Ser Gly Gly Ala 225 230 235 240 Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gly Gly Ala Pro Ser Ser Gln 245 250 255 Tyr Gly Ala Pro Ala Gly Gly Ala Pro Ser Ser Gln Tyr Gly Ala Pro 260 265 270 Ala Gly Gly Ala Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gly Gly Ala 275 280 285 Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gly Gly Ala Pro Ser Ser Gln 290 295 300 Tyr Gly Ala Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gly Gly Ala Pro 305 310 315 320 Ser Ser Gln Tyr Gly Ala Pro Ala Gly Gly Ala Pro Ser Ser Gln Tyr 325 330 335 Gly Ala Pro Ser Gly Gly Ala Pro Ser Ser Gln Tyr Gly Ala Pro Ser 340 345 350 Gly Gly Ala Pro Ser Ser Gln Tyr Gly Ala Pro Ala Gly Gly Ala Pro 355 360 365 Ser Ser Gln Tyr Gly Ala Pro Ser Gly Gly Ala Pro Ser Ser 370 375 380 <210> 33 <211> 491 <212> PRT <213> Bactrocera cucurbitae <400> 33 Arg Pro Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Ser Ala Asn Gly 1 5 10 15 Asn Gly Asn Gly Gly Gly Arg Pro Ser Ser Gln Tyr Gly Ala Pro Gly 20 25 30 Leu Gly Ser Asn Ser Asn Gly Asn Gly Asn Gly Asn Gly Gly Gly Arg 35 40 45 Pro Ser Ser Gln Tyr Gly Val Pro Gly Leu Gly Gly Asn Gly Asn Gly 50 55 60 Asn Gly Asn Gly Gly Gly Gly Gly Arg Pro Ser Ser Ser Tyr Gly Ala 65 70 75 80 Pro Gly Leu Gly Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly Gly Arg 85 90 95 Pro Ser Ser Gln Tyr Gly Val Pro Gly Leu Gly Gly Asn Gly Asn Gly 100 105 110 Asn Gly Asn Gly Asn Gly Gly Gly Arg Pro Ser Ser Thr Tyr Gly Ala 115 120 125 Pro Gly Leu Arg Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Arg 130 135 140 Pro Ser Ser Thr Tyr Gly Ala Pro Gly Leu Gly Gly Asn Gly Asn Gly 145 150 155 160 Asn Gly Asn Gly Asn Gly Arg Pro Ser Ser Thr Tyr Gly Ala Pro Gly 165 170 175 Leu Gly Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Arg Pro Ser 180 185 190 Ser Thr Tyr Gly Ala Pro Gly Leu Asn Gly Asn Gly Leu Gly Gly Gly 195 200 205 Gln Lys Pro Ser Asp Ser Tyr Gly Pro Pro Ala Ser Gly Asn Gly Asn 210 215 220 Gly Tyr Ser Asn Gly Gly Asn Gly Asn Gly Asn Gly Gly Gly Arg Pro 225 230 235 240 Gly Gln Glu Tyr Leu Pro Pro Gly Arg Asn Gly Asn Gly Asn Gly Asn 245 250 255 Gly Gly Arg Gly Asn Gly Asn Gly Gly Gly Ala Asn Gly Tyr Asp Tyr 260 265 270 Ser Gln Gly Gly Ser Asp Ser Gly Glu Ser Gly Ile Val Asp Tyr Glu 275 280 285 Ala Asp Gln Gly Gly Tyr Arg Pro Gln Ile Arg Tyr Glu Gly Glu Ala 290 295 300 Asn Asn Gly Ala Gly Gly Leu Gly Gly Gly Ala Gly Gly Ala Asn Gly 305 310 315 320 Tyr Asp Tyr Glu Gln Asn Gly Asn Gly Leu Gly Gly Gly Asn Gly Tyr 325 330 335 Ser Asn Gly Gln Asp Leu Gly Ser Asn Gly Tyr Ser Ser Gly Arg Pro 340 345 350 Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Tyr Ser 355 360 365 Gly Arg Asn Gly Lys Gly Arg Asn Gly Asn Gly Gly Gly Gln Gly Leu 370 375 380 Gly Arg Asn Gly Tyr Ser Asp Gly Arg Pro Ser Gly Gln Asp Leu Gly 385 390 395 400 Asp Asn Gly Tyr Ala Ser Gly Arg Pro Gly Gly Asn Gly Asn Gly Asn 405 410 415 Gly Gly Asn Gly Asn Gly Tyr Ser Asn Gly Asn Gly Tyr Ser Asn Gly 420 425 430 Asn Gly Asn Gly Thr Gly Asn Gly Gly Gly Gln Tyr Asn Gly Asn Gly 435 440 445 Asn Gly Tyr Ser Asp Gly Arg Pro Gly Gly Gln Asp Asn Leu Asp Gly 450 455 460 Gln Gly Tyr Ser Ser Gly Arg Pro Asn Gly Phe Gly Pro Gly Gly Gln 465 470 475 480 Asn Gly Asp Asn Asp Gly Asn Gly Tyr Arg Tyr 485 490 <210> 34 <211> 770 <212> PRT <213> Trichogramma pretiosum <400> 34 Arg Pro Glu Pro Pro Val Asn Ser Tyr Leu Pro Pro Gly Gln Gly Gly 1 5 10 15 Gln Gly Gly Phe Gly Gly Ser Gly Gly Arg Pro Gly Gly Gly Ser Pro 20 25 30 Ser Asn Gln Tyr Gly Pro Pro Asn Phe Gln Asn Gly Gly Gly Gln Asn 35 40 45 Gly Gly Ser Gly Phe Gly Gly Asn Gly Asn Gly Asn Ser Phe Gly Pro 50 55 60 Pro Ser Asn Ser Tyr Gly Pro Pro Glu Phe Gly Ser Pro Gly Ala Gly 65 70 75 80 Ser Phe Gly Gly Gly Arg Pro Gln Asp Thr Tyr Gly Pro Pro Ser Asn 85 90 95 Gly Asn Gly Asn Gly Asn Gly Phe Gly Gly Asn Gly Asn Gly Gly Gly 100 105 110 Arg Pro Ser Ser Arg Pro Ser Asp Ser Tyr Gly Pro Pro Ser Ser Gly 115 120 125 Asn Gly Phe Gly Gly Gly Asn Ser Gly Arg Pro Ser Glu Ser Tyr Gly 130 135 140 Pro Pro Gln Asn Gly Gly Gly Ser Gly Asn Gly Asn Gln Gly Gly Gly 145 150 155 160 Asn Gly Phe Gly Asn Gly Gly Gly Arg Gly Gly Gln Gly Lys Pro Ser 165 170 175 Asp Ser Tyr Gly Pro Pro Asn Ser Gly Asn Arg Pro Gly Ser Ser Asn 180 185 190 Gly Gly Gly Gln Gln Gln Asn Gly Phe Gly Gly Gly Asn Gly Gly Arg 195 200 205 Pro Ser Asn Thr Tyr Gly Pro Pro Gly Gly Gly Asn Gly Gly Gly Arg 210 215 220 Pro Gly Gly Ser Ser Gly Gly Phe Gly Gly Gln Asn Gly Gly Arg Pro 225 230 235 240 Ser Asp Ser Tyr Gly Pro Pro Ser Asn Gly Asn Gly Asn Gly Gly Arg 245 250 255 Pro Ser Asn Asn Tyr Gly Pro Pro Asn Ser Gly Gly Gly Asn Gly Asn 260 265 270 Gly Phe Gly Gly Ser Asn Gly Lys Pro Ser Asn Ser Tyr Gly Pro Pro 275 280 285 Ser Asn Gly Asn Gly Gly Gly Phe Gly Gly Ser Asn Gly Arg Pro Ser 290 295 300 Asn Ser Tyr Gly Pro Pro Ser Gly Gly Asn Gly Gly Gly Phe Gly Gly 305 310 315 320 Ser Ser Ala Val Gly Arg Pro Gly Asn Ser Gly Ser Pro Ser Ser Ser 325 330 335 Gly Ser Gly Phe Gly Gly Asn Gly Gly Ala Ser Arg Pro Ser Ser Ser 340 345 350 Tyr Gly Pro Pro Ser Asn Gly Gly Gly Phe Gly Asn Gly Gly Gly Ser 355 360 365 Asn Gly Arg Pro Ser Ser Ser Tyr Gly Pro Pro Asn Ser Gly Ser Asn 370 375 380 Gly Gly Gly Phe Gly Gly Gln Asn Gly Asn Gly Arg Gln Asn Gly Asn 385 390 395 400 Asn Gly Gln Gly Gly Phe Gly Gly Gln Pro Ser Ser Ser Tyr Gly Pro 405 410 415 Pro Ser Asn Gly Asn Gly Phe Gly Gly Gly Gly Gly Ser Asn Gly Tyr 420 425 430 Pro Gln Asn Ser Gln Gly Gly Asn Gly Asn Gly Phe Gly Gln Gly Ser 435 440 445 Gly Gly Arg Pro Ser Ser Ser Tyr Gly Pro Pro Ser Asn Gly Gly Gly 450 455 460 Gly Gly Asp Asn Gly Tyr Ser Ser Gly Gly Pro Gly Gly Phe Gly Gly 465 470 475 480 Gln Pro Gln Asp Ser Tyr Gly Pro Pro Pro Ser Gly Ala Val Asp Gly 485 490 495 Asn Asn Gly Phe Ser Ser Gly Gly Ser Ser Gly Asp Asn Asn Gly Tyr 500 505 510 Ser Ser Gly Gly Pro Gly Gly Asn Gly Phe Glu Asp Gly Asn Asp Glu 515 520 525 Pro Ala Lys Tyr Glu Phe Ser Tyr Glu Val Lys Asp Glu Gln Ser Gly 530 535 540 Ser Ser Phe Gly His Thr Glu Met Arg Asp Gly Asp Arg Ala Gln Gly 545 550 555 560 Glu Phe Asn Val Leu Leu Pro Asp Gly Arg Lys Gln Ile Val Glu Tyr 565 570 575 Glu Ala Asp Gln Asp Gly Phe Lys Pro Gln Ile Arg Tyr Glu Gly Glu 580 585 590 Ala Asn Thr Gly Gly Ala Gly Gly Tyr Pro Ser Gly Gly Pro Gly Gly 595 600 605 Gln Gly Gly Asn Gly Asn Gly Gly Tyr Pro Ser Gly Gly Pro Ser Asn 610 615 620 Gly Gly Phe Gly Gly Gln Asn Gly Gly Gly Asn Gly Gly Tyr Pro Ser 625 630 635 640 Gly Gly Pro Ser Gly Gly Gly Phe Gly Gly Gln Asn Gly Gly Ser Gly 645 650 655 Gly Tyr Pro Ser Gly Gly Pro Ser Gly Gly Gly Phe Gly Gly Gln Gly 660 665 670 Gly Phe Gly Gly Gln Asn Ser Gly Gly Asn Gly Gly Tyr Ser Ser Gly 675 680 685 Gly Pro Ala Ser Gly Gly Phe Gly Gly Gln Asn Gly Gly Asn Gly Gly 690 695 700 Tyr Pro Ser Gly Gly Pro Ser Gly Gly Gly Phe Gly Gly Gln Gly Gly 705 710 715 720 Phe Gly Gly Gln Asn Ser Gly Gly Asn Gly Gly Tyr Pro Ser Gly Gly 725 730 735 Pro Ser Ser Gly Gly Phe Gly Gly Gln Asn Gly Gly Gly Gly Gly Asn 740 745 750 Tyr Pro Ala Gly Ser Gly Gly Asp Ala Glu Ala Asn Gly Gly Tyr Gln 755 760 765 Tyr Ser 770 <210> 35 <211> 280 <212> PRT <213> Drosophila sechellia <400> 35 Gln Ser Gly Ala Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly 1 5 10 15 Gly Gly Asn Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln 20 25 30 Gly Gln Gly Gln Gly Gln Gly Gln Gly Gly Tyr Gly Gly Lys Pro Ser 35 40 45 Asp Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro 50 55 60 Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser 65 70 75 80 Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp 85 90 95 Thr Tyr Gly Ala Pro Gly Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro 100 105 110 Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Asn Gly Asn Gly 115 120 125 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly 130 135 140 Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg 145 150 155 160 Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Asn Gly Gly Arg 165 170 175 Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro 180 185 190 Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Ser Gly 195 200 205 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ala Gln Gly Gln Gly Gly 210 215 220 Phe Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln Asn Gln 225 230 235 240 Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly Asn Gly Ser Ala 245 250 255 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Gly Pro Gly Gly 260 265 270 Arg Pro Ser Asp Ser Tyr Gly Pro 275 280 <210> 36 <211> 280 <212> PRT <213> Drosophila sechellia <400> 36 Gln Ser Gly Ala Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly 1 5 10 15 Gly Gly Asn Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln 20 25 30 Gly Gln Gly Gln Gly Gln Gly Gln Gly Gly Tyr Gly Gly Lys Pro Ser 35 40 45 Asp Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro 50 55 60 Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser 65 70 75 80 Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp 85 90 95 Thr Tyr Gly Ala Pro Gly Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro 100 105 110 Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Asn Gly Asn Gly 115 120 125 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly 130 135 140 Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg 145 150 155 160 Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Asn Gly Gly Arg 165 170 175 Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro 180 185 190 Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Ser Gly 195 200 205 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ala Gln Gly Gln Gly Gly 210 215 220 Phe Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln Asn Gln 225 230 235 240 Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly Asn Gly Ser Ala 245 250 255 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Gly Pro Gly Gly 260 265 270 Arg Pro Ser Asp Ser Tyr Gly Pro 275 280 <210> 37 <211> 280 <212> PRT <213> Drosophila sechellia <400> 37 Gln Ser Gly Ala Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly 1 5 10 15 Gly Gly Asn Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln 20 25 30 Gly Gln Gly Gln Gly Gln Gly Gln Gly Gly Tyr Gly Gly Lys Pro Ser 35 40 45 Asp Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro 50 55 60 Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser 65 70 75 80 Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp 85 90 95 Thr Tyr Gly Ala Pro Gly Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro 100 105 110 Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Asn Gly Asn Gly 115 120 125 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly 130 135 140 Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg 145 150 155 160 Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Asn Gly Gly Arg 165 170 175 Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro 180 185 190 Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Ser Gly 195 200 205 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ala Gln Gly Gln Gly Gly 210 215 220 Phe Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln Asn Gln 225 230 235 240 Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly Asn Gly Ser Ala 245 250 255 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Gly Pro Gly Gly 260 265 270 Arg Pro Ser Asp Ser Tyr Gly Pro 275 280 <210> 38 <211> 280 <212> PRT <213> Drosophila sechellia <400> 38 Gln Ser Gly Ala Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly 1 5 10 15 Gly Gly Asn Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln 20 25 30 Gly Gln Gly Gln Gly Gln Gly Gln Gly Gly Tyr Gly Gly Lys Pro Ser 35 40 45 Asp Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro 50 55 60 Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser 65 70 75 80 Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp 85 90 95 Thr Tyr Gly Ala Pro Gly Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro 100 105 110 Ser Ser Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Asn Gly Asn Gly 115 120 125 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly 130 135 140 Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg 145 150 155 160 Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Asn Gly Gly Arg 165 170 175 Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro 180 185 190 Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Ser Gly 195 200 205 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Ala Gln Gly Gln Gly Gly 210 215 220 Phe Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln Asn Gln 225 230 235 240 Lys Pro Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly Asn Gly Ser Ala 245 250 255 Gly Arg Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Gly Pro Gly Gly 260 265 270 Arg Pro Ser Asp Ser Tyr Gly Pro 275 280 <210> 39 <211> 162 <212> PRT <213> Drosophila sechellia <400> 39 Tyr Ser Ser Gly Arg Pro Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly 1 5 10 15 Tyr Ser Ser Gly Arg Pro Gly Gly Gln Asp Leu Gly Pro Ser Gly Tyr 20 25 30 Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Gly Tyr Ser 35 40 45 Asn Val Lys Pro Gly Gly Gln Asp Leu Gly Pro Gly Gly Tyr Ser Gly 50 55 60 Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Gly Gly 65 70 75 80 Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Ala Tyr Ser Asn Gly Arg 85 90 95 Pro Gly Gly Asn Gly Asn Gly Gly Ser Asp Gly Gly Arg Val Ile Ile 100 105 110 Gly Gly Arg Val Ile Gly Gly Gln Asp Gly Gly Asp Gln Gly Tyr Ser 115 120 125 Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Ser 130 135 140 Gly Arg Pro Gly Gly Arg Pro Gly Gly Asn Gly Gln Asp Ser Gln Asp 145 150 155 160 Gly Gln <210> 40 <211> 162 <212> PRT <213> Drosophila sechellia <400> 40 Tyr Ser Ser Gly Arg Pro Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly 1 5 10 15 Tyr Ser Ser Gly Arg Pro Gly Gly Gln Asp Leu Gly Pro Ser Gly Tyr 20 25 30 Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Gly Tyr Ser 35 40 45 Asn Val Lys Pro Gly Gly Gln Asp Leu Gly Pro Gly Gly Tyr Ser Gly 50 55 60 Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Gly Gly 65 70 75 80 Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Ala Tyr Ser Asn Gly Arg 85 90 95 Pro Gly Gly Asn Gly Asn Gly Gly Ser Asp Gly Gly Arg Val Ile Ile 100 105 110 Gly Gly Arg Val Ile Gly Gly Gln Asp Gly Gly Asp Gln Gly Tyr Ser 115 120 125 Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Ser 130 135 140 Gly Arg Pro Gly Gly Arg Pro Gly Gly Asn Gly Gln Asp Ser Gln Asp 145 150 155 160 Gly Gln <210> 41 <211> 162 <212> PRT <213> Drosophila sechellia <400> 41 Tyr Ser Ser Gly Arg Pro Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly 1 5 10 15 Tyr Ser Ser Gly Arg Pro Gly Gly Gln Asp Leu Gly Pro Ser Gly Tyr 20 25 30 Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Gly Tyr Ser 35 40 45 Asn Val Lys Pro Gly Gly Gln Asp Leu Gly Pro Gly Gly Tyr Ser Gly 50 55 60 Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Gly Gly 65 70 75 80 Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Ala Tyr Ser Asn Gly Arg 85 90 95 Pro Gly Gly Asn Gly Asn Gly Gly Ser Asp Gly Gly Arg Val Ile Ile 100 105 110 Gly Gly Arg Val Ile Gly Gly Gln Asp Gly Gly Asp Gln Gly Tyr Ser 115 120 125 Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Ser 130 135 140 Gly Arg Pro Gly Gly Arg Pro Gly Gly Asn Gly Gln Asp Ser Gln Asp 145 150 155 160 Gly Gln <210> 42 <211> 162 <212> PRT <213> Drosophila sechellia <400> 42 Tyr Ser Ser Gly Arg Pro Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly 1 5 10 15 Tyr Ser Ser Gly Arg Pro Gly Gly Gln Asp Leu Gly Pro Ser Gly Tyr 20 25 30 Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Gly Tyr Ser 35 40 45 Asn Val Lys Pro Gly Gly Gln Asp Leu Gly Pro Gly Gly Tyr Ser Gly 50 55 60 Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Gly Gly 65 70 75 80 Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Ala Tyr Ser Asn Gly Arg 85 90 95 Pro Gly Gly Asn Gly Asn Gly Gly Ser Asp Gly Gly Arg Val Ile Ile 100 105 110 Gly Gly Arg Val Ile Gly Gly Gln Asp Gly Gly Asp Gln Gly Tyr Ser 115 120 125 Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Ser 130 135 140 Gly Arg Pro Gly Gly Arg Pro Gly Gly Asn Gly Gln Asp Ser Gln Asp 145 150 155 160 Gly Gln <210> 43 <211> 162 <212> PRT <213> Drosophila sechellia <400> 43 Tyr Ser Ser Gly Arg Pro Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly 1 5 10 15 Tyr Ser Ser Gly Arg Pro Gly Gly Gln Asp Leu Gly Pro Ser Gly Tyr 20 25 30 Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Gly Tyr Ser 35 40 45 Asn Val Lys Pro Gly Gly Gln Asp Leu Gly Pro Gly Gly Tyr Ser Gly 50 55 60 Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Gly Gly 65 70 75 80 Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Ala Tyr Ser Asn Gly Arg 85 90 95 Pro Gly Gly Asn Gly Asn Gly Gly Ser Asp Gly Gly Arg Val Ile Ile 100 105 110 Gly Gly Arg Val Ile Gly Gly Gln Asp Gly Gly Asp Gln Gly Tyr Ser 115 120 125 Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Ser 130 135 140 Gly Arg Pro Gly Gly Arg Pro Gly Gly Asn Gly Gln Asp Ser Gln Asp 145 150 155 160 Gly Gln <210> 44 <211> 162 <212> PRT <213> Drosophila sechellia <400> 44 Tyr Ser Ser Gly Arg Pro Gly Asn Gly Asn Gly Asn Gly Asn Gly Gly 1 5 10 15 Tyr Ser Ser Gly Arg Pro Gly Gly Gln Asp Leu Gly Pro Ser Gly Tyr 20 25 30 Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Gly Tyr Ser 35 40 45 Asn Val Lys Pro Gly Gly Gln Asp Leu Gly Pro Gly Gly Tyr Ser Gly 50 55 60 Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Gly Gly 65 70 75 80 Arg Pro Gly Gly Gln Asp Leu Gly Ala Gly Ala Tyr Ser Asn Gly Arg 85 90 95 Pro Gly Gly Asn Gly Asn Gly Gly Ser Asp Gly Gly Arg Val Ile Ile 100 105 110 Gly Gly Arg Val Ile Gly Gly Gln Asp Gly Gly Asp Gln Gly Tyr Ser 115 120 125 Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Arg Asp Gly Tyr Ser Ser 130 135 140 Gly Arg Pro Gly Gly Arg Pro Gly Gly Asn Gly Gln Asp Ser Gln Asp 145 150 155 160 Gly Gln <210> 45 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 Gly Asp Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp 1 5 10 15 Lys Asp Tyr Lys Asp Asp Asp Asp Lys 20 25 <210> 46 <211> 85 <212> PRT <213> Unknown <220> <223> Description of Unknown: Alpha mating factor precursor protein sequence <400> 46 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Glu Lys Arg 85 <210> 47 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Glu Ala Glu Ala 1 <210> 48 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Ser Gly 1 <210> 49 <211> 722 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 49 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Glu Lys Arg Glu Ala Glu Ala Gly Arg Pro Glu Pro Pro Val 85 90 95 Asn Ser Tyr Leu Pro Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln Ser 100 105 110 Gly Ala Gly Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly 115 120 125 Asn Gly Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln Gly Gln 130 135 140 Gly Gln Gly Gln Gly Gln Gly Gly Tyr Gly Gly Lys Pro Ser Asp Ser 145 150 155 160 Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro Ser Ser 165 170 175 Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp Thr 180 185 190 Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp Thr Tyr 195 200 205 Gly Ala Pro Gly Gly Gly Gly Asn Gly Asn Gly Gly Arg Pro Ser Ser 210 215 220 Ser Tyr Gly Ala Pro Gly Gln Gly Gln Gly Asn Gly Asn Gly Gly Arg 225 230 235 240 Pro Ser Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro 245 250 255 Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser 260 265 270 Asp Thr Tyr Gly Ala Pro Gly Gly Gly Asn Asn Gly Gly Arg Pro Ser 275 280 285 Ser Ser Tyr Gly Ala Pro Gly Gly Gly Asn Gly Gly Arg Pro Ser Asp 290 295 300 Thr Tyr Gly Ala Pro Gly Gly Gly Asn Gly Asn Gly Ser Gly Gly Arg 305 310 315 320 Pro Ser Ser Ser Tyr Gly Ala Pro Ala Gln Gly Gln Gly Gly Phe Gly 325 330 335 Gly Arg Pro Ser Asp Ser Tyr Gly Ala Pro Gly Gln Asn Gln Lys Pro 340 345 350 Ser Asp Ser Tyr Gly Ala Pro Gly Ser Gly Asn Gly Ser Ala Gly Arg 355 360 365 Pro Ser Ser Ser Tyr Gly Ala Pro Gly Ser Gly Pro Gly Gly Arg Pro 370 375 380 Ser Asp Ser Tyr Gly Pro Pro Ala Ser Gly Ser Gly Ala Gly Gly Ala 385 390 395 400 Gly Gly Ser Gly Pro Gly Gly Ala Asp Tyr Asp Asn Asp Glu Pro Ala 405 410 415 Lys Tyr Glu Phe Asn Tyr Gln Val Glu Asp Ala Pro Ser Gly Leu Ser 420 425 430 Phe Gly His Ser Glu Met Arg Asp Gly Asp Phe Thr Thr Gly Gln Tyr 435 440 445 Asn Val Leu Leu Pro Asp Gly Arg Lys Gln Ile Val Glu Tyr Glu Ala 450 455 460 Asp Gln Gln Gly Tyr Arg Pro Gln Ile Arg Tyr Glu Gly Asp Ala Asn 465 470 475 480 Asp Gly Ser Gly Pro Ser Gly Pro Ser Gly Pro Gly Gly Pro Gly Gly 485 490 495 Gln Asn Leu Gly Ala Asp Gly Tyr Ser Ser Gly Arg Pro Gly Asn Gly 500 505 510 Asn Gly Asn Gly Asn Gly Gly Tyr Ser Ser Gly Arg Pro Gly Gly Gln 515 520 525 Asp Leu Gly Pro Ser Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp 530 535 540 Leu Gly Ala Gly Gly Tyr Ser Asn Val Lys Pro Gly Gly Gln Asp Leu 545 550 555 560 Gly Pro Gly Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly 565 570 575 Arg Asp Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu Gly Ala 580 585 590 Gly Ala Tyr Ser Asn Gly Arg Pro Gly Gly Asn Gly Asn Gly Gly Ser 595 600 605 Asp Gly Gly Arg Val Ile Ile Gly Gly Arg Val Ile Gly Gly Gln Asp 610 615 620 Gly Gly Asp Gln Gly Tyr Ser Gly Gly Arg Pro Gly Gly Gln Asp Leu 625 630 635 640 Gly Arg Asp Gly Tyr Ser Ser Gly Arg Pro Gly Gly Arg Pro Gly Gly 645 650 655 Asn Gly Gln Asp Ser Gln Asp Gly Gln Gly Tyr Ser Ser Gly Arg Pro 660 665 670 Gly Gln Gly Gly Arg Asn Gly Phe Gly Pro Gly Gly Gln Asn Gly Asp 675 680 685 Asn Asp Gly Ser Gly Tyr Arg Tyr Ser Gly Asp Tyr Lys Asp Asp Asp 690 695 700 Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp 705 710 715 720 Asp Lys <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(3) <223> Any amino acid <220> <221> MOD_RES <222> (6)..(6) <223> Any amino acid <400> 50 Ser Xaa Xaa Tyr Gly Xaa Pro 1 5 <210> 51 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 51 Gly Gly Gly Gly 1 <210> 52 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Asn Gly Asn Gly 1 <210> 53 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Gly Gln Gly Gly 1 <210> 54 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Gly Gln Gly Asn 1 <210> 55 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 55 Gly Gln Gly Gln 1 <210> 56 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 56 Gly Gln Gly Gln Gly 1 5 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Arg Pro Gly Gly Gln 1 5 <210> 58 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Arg Pro Gly Gly Asn 1 5 <210> 59 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Gly Gly Ser Phe 1 <210> 60 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Gly Asn Gly Gly 1 <210> 61 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 61 Gly Gly Ala Gly Gly 1 5 <210> 62 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (1)…(2) <223> Any amino acid <400> 62 Yes Yes Glu Pro Pro Val Ser Tyr Leu Pro Pro Ser 1 5 10 <210> 63 <211> 4 <212> PRT <213> Unknown <220> <223> Description of Unknown: "GRPE" sequence <400> 63 Gly Arg Pro Glu 1 <210> 64 <211> 3000 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURES <222> (1)..(3000) <223> This sequence may encompass 1-100″ Tyr Gly Xaa Pro Xaa Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes” repeating units <220> <221> MOD_RES <222> (3)…(3) <223> A, L, P, T or V <220> <221> MOD_RES <222> (5)..(24) <223> Any amino acid <220> <221> MISC_FEATURE <222> (5)..(24) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (25)..(28) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (29)..(30) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (33)..(33) <223> A, L, P, T or V <220> <221> MOD_RES <222> (35)..(54) <223> Any amino acid <220> <221> MISC_FEATURE <222> (35)..(54) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (55)..(58) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (59)..(60) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (63)..(63) <223> A, L, P, T or V <220> <221> MOD_RES <222> (65)..(84) <223> Any amino acid <220> <221> MISC_FEATURE <222> (65)..(84) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (85)..(88) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (89)..(90) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (93)..(93) <223> A, L, P, T or V <220> <221> MOD_RES <222> (95)..(114) <223> Any amino acid <220> <221> MISC_FEATURE <222> (95)..(114) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (115)..(118) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (119)..(120) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (123)..(123) <223> A, L, P, T or V <220> <221> MOD_RES <222> (125)..(144) <223> Any amino acid <220> <221> MISC_FEATURE <222> (125)..(144) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (145)..(148) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (149)..(150) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (153)..(153) <223> A, L, P, T or V <220> <221> MOD_RES <222> (155)..(174) <223> Any amino acid <220> <221> MISC_FEATURE <222> (155)..(174) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (175)..(178) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (179)..(180) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (183)..(183) <223> A, L, P, T or V <220> <221> MOD_RES <222> (185)..(204) <223> Any amino acid <220> <221> MISC_FEATURE <222> (185)..(204) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (205)..(208) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (209)..(210) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (213)..(213) <223> A, L, P, T or V <220> <221> MOD_RES <222> (215)..(234) <223> Any amino acid <220> <221> MISC_FEATURE <222> (215)..(234) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (235)..(238) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (239)..(240) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (243)..(243) <223> A, L, P, T or V <220> <221> MOD_RES <222> (245)..(264) <223> Any amino acid <220> <221> MISC_FEATURE <222> (245)..(264) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (265)..(268) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (269)..(270) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (273)..(273) <223> A, L, P, T or V <220> <221> MOD_RES <222> (275)..(294) <223> Any amino acid <220> <221> MISC_FEATURE <222> (275)..(294) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (295)..(298) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (299)..(300) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (303)..(303) <223> A, L, P, T or V <220> <221> MOD_RES <222> (305)..(324) <223> Any amino acid <220> <221> MISC_FEATURE <222> (305)..(324) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (325)..(328) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (329)..(330) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (333)..(333) <223> A, L, P, T or V <220> <221> MOD_RES <222> (335)..(354) <223> Any amino acid <220> <221> MISC_FEATURE <222> (335)..(354) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (355)..(358) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (359)..(360) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (363)..(363) <223> A, L, P, T or V <220> <221> MOD_RES <222> (365)..(384) <223> Any amino acid <220> <221> MISC_FEATURE <222> (365)..(384) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (385)..(388) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (389)..(390) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (393)..(393) <223> A, L, P, T or V <220> <221> MOD_RES <222> (395)..(414) <223> Any amino acid <220> <221> MISC_FEATURE <222> (395)..(414) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (415)..(418) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (419)..(420) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (423)..(423) <223> A, L, P, T or V <220> <221> MOD_RES <222> (425)..(444) <223> Any amino acid <220> <221> MISC_FEATURE <222> (425)..(444) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (445)..(448) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (449)..(450) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (453)..(453) <223> A, L, P, T or V <220> <221> MOD_RES <222> (455)..(474) <223> Any amino acid <220> <221> MISC_FEATURE <222> (455)..(474) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (475)..(478) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (479)..(480) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (483)..(483) <223> A, L, P, T or V <220> <221> MOD_RES <222> (485)..(504) <223> Any amino acid <220> <221> MISC_FEATURE <222> (485)..(504) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (505)..(508) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (509)..(510) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (513)..(513) <223> A, L, P, T or V <220> <221> MOD_RES <222> (515)..(534) <223> Any amino acid <220> <221> MISC_FEATURE <222> (515)..(534) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (535)..(538) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (539)..(540) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (543)..(543) <223> A, L, P, T or V <220> <221> MOD_RES <222> (545)..(564) <223> Any amino acid <220> <221> MISC_FEATURE <222> (545)..(564) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (565)..(568) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (569)..(570) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (573)..(573) <223> A, L, P, T or V <220> <221> MOD_RES <222> (575)..(594) <223> Any amino acid <220> <221> MISC_FEATURE <222> (575)..(594) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (595)..(598) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (599)..(600) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (603)..(603) <223> A, L, P, T or V <220> <221> MOD_RES <222> (605)..(624) <223> Any amino acid <220> <221> MISC_FEATURE <222> (605)..(624) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (625)..(628) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (629)..(630) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (633)..(633) <223> A, L, P, T or V <220> <221> MOD_RES <222> (635)..(654) <223> Any amino acid <220> <221> MISC_FEATURE <222> (635)..(654) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (655)..(658) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (659)..(660) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (663)..(663) <223> A, L, P, T or V <220> <221> MOD_RES <222> (665)..(684) <223> Any amino acid <220> <221> MISC_FEATURE <222> (665)..(684) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (685)..(688) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (689)..(690) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (693)..(693) <223> A, L, P, T or V <220> <221> MOD_RES <222> (695)..(714) <223> Any amino acid <220> <221> MISC_FEATURE <222> (695)..(714) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (715)..(718) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (719)..(720) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (723)..(723) <223> A, L, P, T or V <220> <221> MOD_RES <222> (725)..(744) <223> Any amino acid <220> <221> MISC_FEATURE <222> (725)..(744) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (745)..(748) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (749)..(750) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (753)..(753) <223> A, L, P, T or V <220> <221> MOD_RES <222> (755)..(774) <223> Any amino acid <220> <221> MISC_FEATURE <222> (755)..(774) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (775)..(778) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (779)..(780) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (783)..(783) <223> A, L, P, T or V <220> <221> MOD_RES <222> (785)..(804) <223> Any amino acid <220> <221> MISC_FEATURE <222> (785)..(804) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (805)..(808) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (809)..(810) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (813)..(813) <223> A, L, P, T or V <220> <221> MOD_RES <222> (815)..(834) <223> Any amino acid <220> <221> MISC_FEATURE <222> (815)..(834) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (835)..(838) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (839)..(840) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (843)..(843) <223> A, L, P, T or V <220> <221> MOD_RES <222> (845)..(864) <223> Any amino acid <220> <221> MISC_FEATURE <222> (845)..(864) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (865)..(868) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (869)..(870) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (873)..(873) <223> A, L, P, T or V <220> <221> MOD_RES <222> (875)..(894) <223> Any amino acid <220> <221> MISC_FEATURE <222> (875)..(894) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (895)..(898) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (899)..(900) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (903)..(903) <223> A, L, P, T or V <220> <221> MOD_RES <222> (905)..(924) <223> Any amino acid <220> <221> MISC_FEATURE <222> (905)..(924) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (925)..(928) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (929)..(930) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (933)..(933) <223> A, L, P, T or V <220> <221> MOD_RES <222> (935)..(954) <223> Any amino acid <220> <221> MISC_FEATURE <222> (935)..(954) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (955)..(958) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (959)..(960) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (963)..(963) <223> A, L, P, T or V <220> <221> MOD_RES <222> (965)..(984) <223> Any amino acid <220> <221> MISC_FEATURE <222> (965)..(984) <223> This region may encompass 3-20 residues <220> <221> MOD_RES <222> (985)..(988) <223> This region may encompass one of the following sequences: GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG <220> <221> MOD_RES <222> (989)..(990) <223> This region may encompass one of the following sequences: S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS <220> <221> MOD_RES <222> (993)..(993) <223> A, L, P, T or V <220> <221> MOD_RES <222> (995)..(1014) <223> Any amino acid <220> <221> MISC_FEATURE ...

Claims

1. Providing a composition comprising purified recombinant resilin; placing the recombinant resilin in a cross-linking solution containing ammonium persulfate; and incubating said recombinant resilin in said crosslinking solution at a temperature of at least 60° C., thereby producing a crosslinked recombinant resilin solid composition. A method for crosslinking recombinant resilin comprising:

2. 10. The method of claim 1, wherein the incubation is carried out for at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, or at least 2 hours.

3. 2. The method of any one of the preceding claims, wherein the recombinant resilin in the crosslinking solution is incubated at a temperature of 60°C to 85°C, 70°C to 85°C, or 75°C to 85°C.

4. 2. The method according to any one of the preceding claims, wherein the incubation is carried out for at least 2 hours.

5. 2. The method of any one of the preceding claims, wherein the cross-linking solution does not contain a photocatalyst or a cross-linking enzyme.

6. 2. The method of any one of the preceding claims, wherein the crosslinked recombinant resilin solid composition is stable at room temperature for more than 5 days, more than 10 days, more than 20 days, or more than 40 days.

7. 2. The method of any one of the preceding claims, wherein the purified recombinant resilin is prepared by recombinantly expressing a gene encoding the recombinant resilin in a modified organism in culture and purifying the expressed recombinant resilin from the culture.

8. A composition comprising cross-linked recombinant resilin, wherein the recombinant resilin is cross-linked by exposing the resilin to ammonium persulfate and heat.

9. A composition comprising cross-linked recombinant resilin, the composition being free of a cross-linking enzyme.

10. A composition comprising cross-linked recombinant resilin, the composition being free of a photocatalyst.

11. A recombinant resilin composition comprising cross-linked recombinant resilin in a polar non-aqueous solvent.

12. The composition of claim 11 , wherein the polar non-aqueous solvent is a protic solvent.

13. 13. The composition of claim 12, wherein the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol.

14. The composition of claim 11 , wherein the polar non-aqueous solvent is an aprotic solvent.

15. The composition of claim 14 , wherein the aprotic solvent is DMSO.

16. A composition according to any one of claims 11 to 15, comprising 20 to 40% by weight of resilin.

17. 17. The composition of any one of claims 11 to 16, wherein the resilin is at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a fraction of total resilin as measured by size exclusion chromatography.

18. 18. The composition of any one of claims 11 to 17, wherein the polar non-aqueous solvent is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% by volume of the solvent.

19. The composition of any one of claims 11 to 18, wherein the recombinant resilin composition has a greater elastic modulus than a similar cross-linked recombinant resilin in an aqueous medium.

20. 20. The composition of any one of claims 11-19, wherein the recombinant resilin composition comprises a hardness of at least 10 as measured using a Shore OO durometer according to ASTM D2240.

21. 21. The composition of any one of claims 11-20, wherein the recombinant resilin composition comprises a hardness of about 10 to about 50, as measured using a Shore OO durometer according to ASTM D2240.

22. 22. The composition of any one of claims 11-21, wherein the recombinant resilin composition comprises a rebound resilience of about 40% to about 60% as measured by ASTM D7121.

23. 23. The composition of any one of claims 11-22, wherein the recombinant resilin composition comprises a compressive stress at 25% of about 6 to about 8 psi as measured by ASTM D575.

24. 24. The composition of any one of claims 11 to 23, wherein the recombinant resilin composition does not undergo an elastic to plastic transition at a compressive force of less than 2 kN as measured in a Zwick compression test.

25. The composition of any one of claims 11 to 24, wherein the recombinant resilin composition is a foamed material.

26. providing a crosslinked recombinant resilin solid composition in an aqueous solvent; and exchanging the aqueous solvent for a polar non-aqueous solvent. A method for preparing a recombinant resilin solid, comprising:

27. 27. The method of claim 26, wherein the polar non-aqueous solvent is a protic solvent.

28. 28. The method of claim 27, wherein the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol.

29. 27. The method of claim 26, wherein the polar non-aqueous solvent is an aprotic solvent.

30. 30. The method of claim 29, wherein the aprotic solvent is DMSO.

31. 31. The method of any one of claims 26 to 30, wherein the solvent exchange is carried out for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours.

32. The method of any one of claims 26 to 31, wherein the solvent exchange is carried out at about 60°C.

33. 33. The method of any one of claims 26-32, wherein the crosslinked recombinant resilin solid composition comprises at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a fraction of total resilin as measured by size exclusion chromatography.

34. The method of any one of claims 26 to 33, wherein the crosslinked recombinant solid composition is prepared by the method of any one of claims 1 to 7.

35. providing a crosslinked recombinant resilin solid composition comprising an aqueous solvent; and performing a solvent exchange to replace the aqueous solvent with a polar non-aqueous solvent. A method for adjusting the material properties of a solid composition comprising a crosslinked recombinant resilin solid, comprising:

36. 36. The method of claim 35, wherein replacing the solvent changes the elastic modulus, elasticity, hardness, maximum elastic compressive load, or material lifetime of the crosslinked recombinant resilin solid composition.

37. Providing a composition comprising purified recombinant resilin; placing the recombinant resilin in an aqueous solvent containing ammonium persulfate; incubating said recombinant resilin in said aqueous solvent at a temperature of at least 60° C., thereby producing a crosslinked recombinant resilin solid composition; and exchanging the aqueous solvent for a polar non-aqueous solvent selected from the group consisting of glycerol, propylene glycol, ethylene glycol, and DMSO. A method for preparing a recombinant crosslinked resilin solid, comprising:

38. providing a crosslinked recombinant resilin solid composition in an aqueous solvent; exchanging the aqueous solvent for a polar non-aqueous solvent; and introducing one or more gas bubbles into said crosslinked recombinant resilin solid composition. A method for preparing a recombinant resilin foam comprising:

39. 39. The method of claim 38, wherein the polar non-aqueous solvent is a protic solvent.

40. 40. The method of claim 39, wherein the protic solvent is selected from the group consisting of glycerol, propylene glycol, and ethylene glycol.

41. 39. The method of claim 38, wherein the polar non-aqueous solvent is an aprotic solvent.

42. 42. The method of claim 41 , wherein the aprotic solvent is DMSO.

43. 43. The method of any one of claims 38 to 42, wherein the solvent exchange is carried out for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours.

44. 44. The method of any one of claims 38 to 43, wherein the solvent exchange is carried out at about 60°C.

45. 45. The method of any one of claims 38-44, wherein the crosslinked recombinant resilin solid composition comprises at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a fraction of total resilin as measured by size exclusion chromatography.

46. The method of any one of claims 38 to 45, wherein the crosslinked recombinant solid composition is prepared by the method of any one of claims 1 to 7.

47. 47. The method of any one of claims 38-46, wherein said introducing comprises adding a foaming agent to said crosslinked recombinant resilin solid composition.

48. 48. The method of claim 47, wherein the blowing agent comprises a chemical blowing agent.

49. 49. The method of claim 48, wherein the chemical blowing agent comprises sodium bicarbonate, potassium bicarbonate, ammonium, azodicarbonamide, isocyanate, hydrazine, isopropanol, 5-phenyltetrazole, triazole, 4,4'oxybis(benzenesulfonylhydrazide) (OBSH), trihydrazine triazine (THT), hydrogen phosphate, tartaric acid, citric acid, and toluenesulfonyl semicarbazide (TSS).

50. 49. The method of claim 48, wherein the chemical blowing agent comprises sodium bicarbonate.

51. 48. The method of claim 47, wherein the blowing agent comprises a physical blowing agent.

52. The physical blowing agent is a chlorofluorocarbon (CFC), dissolved nitrogen, N ２ , C.H. ４ , H ２ , CO ２ 52. The method of claim 51, wherein the mixture comprises Ar, pentane, isopentane, hexane, methylene dichloride, and dichlorotetrafluoroethane.

53. 52. The method of claim 51 , wherein the physical blowing agent comprises dissolved nitrogen.

54. A shoe insole comprising a composition prepared by the method of any one of claims 1-7, 25-36, and 38-53.

55. A composition comprising a crosslinked resilin composition in a polar non-aqueous solvent, wherein the crosslinked resilin is foamed.

56. 56. The composition of claim 55, wherein the foamed crosslinked resilin comprises at least 50% porosity.

57. 56. The composition of claim 55, wherein the foamed crosslinked resilin has an average cell diameter of from about 0.01 mm to about 3 mm.

58. A shoe insole comprising a composition according to any one of claims 8 to 25 and claims 55 to 57.

Images