USOOS245O12A United States Patent (19) 11 Patent Number: 5,245,012 Lombari et al. 45) Date of Patent: Sep. 14, 1993 (54) METHOD TO ACHIEVE SOLUBILIZATION Work et al., 1982, J. Arachnol, 10:1-10. OF SPIDER SLK PROTEINS Dong et al., 1991, Arch. Biochem. Biophys., 75 Inventors: Stephen J. Lombari, Brighton; David 284(1);53-57. Hall, N., 1988, New Scientist, 29:39. L. Kaplan, Stow, both of Mass. Abstract, Biosir No. 72031529 of Candelas et al., 1981, 73) Assignee: The United States of America as J. Exp. Zool., 216(1):1-6. represented by the Secretary of the Chemical Abstract No. 67:55000p, of Vecchio et al., Army, Washington, D.C. 1967. (21) Appl. No.: 953,323 Chemical Abstract No. 98:199685d of Bhat et al., 1983. 22 Filed: Sep. 29, 1992 Chemical Abstract No. 89:1875p of Sagar et al., 1978. Yuen et al., 1989, Biotechniques, 7(1):74-81. Related U.S. Application Data Xu et al., 1990, Proc. Natl. Acad. Sci., 87:7120-7124. 63) Continuation of Ser. No. 511,114, Apr. 19, 1990, aban Andersen, "Amino Acid Composition of Spider Silk', doned. Comp. Biochem. Physiol, 35:705-711 (1970). 51) Int. Cl......................... C07K 15/20; C07K 3/00; Gosline et al., "Spider Silk as a Rubber', Nature, C07K 15/00; C07K 15/08 309:551-552 (1984). (52) U.S. C. .................................... 530/353; 530/412; Hunt, S., "Amino Acid Composition of Silk from the 530/422; 530/425; 8/127.6; 8/128.1 Pseudoscorpion Neobisium maritimum (Leach): a Possi ble Link between Silk Fibroins and Keratins', Comp. 58) Field of Search ............... 530/353,412,422, 425; Biochem. Physiol, 34:773-776 (1970). 8/127.6, 128.1 Lewis, R. V., "Cloning and Structure of Different (56) References Cited Types of Spider Silk”, DTIC, AD-A203 137. U.S. PATENT DOCUMENTS Lucas et al., "Comparative Studies of Fibroins. The Amino Acid Composition of Various Fibroins and Its Re. 21,454 2/1939 Bly et al. ............................. 530/353 Significance in Relation to their Crystal Structure", 4,394,443 7/1983 Weissman et al. ...................... 435/6 Mol. Biol., 2:339-49 (1960). FOREIGN PATENT DOCUMENTS O'Sullivan, D., "Spider Silk Gene Route to High-ten sile Fiber', Chemical and Engineering News, Jul. 25, 294979 12/1988 European Pat. Off. 1988. 8803533 5/1988 PCT Int'l Appl. , Tillinghast & Christensen, "Observations on the Chemi 2162190 l/1986 United Kingdom. cal Composition of the Web of Nephila clavipes, (Ara OTHER PUBLICATIONS neae, Araneidea)', J. Arachnol, 10:69-74 (1984). Work, R. W. and C. T. Young, 1987, The Amino Acid Zemlan, "A Study of the Mechanical Behavior of Spi Compositions of Major and Minor Ampullate Silks of der Silks", Technical Report 69-29-CM, AD 684333, Certain Orb-Web-Building Spiders (Araneae, Aranei U.S. Army Natick Laboratories, Natick, Mass. dae), J. Arachnol, 15:65-80. 01760-5020 (1968). Abstract entitled: "The Nephila Clavipes Major Am Primary Examiner-Keith C. Furman pullate Gland Characterization of Large Polypeptides, Attorney, Agent, or Firm-Richard J. Donohue Detection of Silk Gene-Related DNA in Nephila Cla vipes Genome", S. J. Lombardi & D. L. Kaplan, XI 57) ABSTRACT International Congress of Arachnology, Turku, Fin A recombinant spider silk protein can be obtained in a land, Aug. 7-12, 1989. commercially useful form by cloning and the expression Abstract, accession No. 81 18626 of DIALOG File 5; in a host cell of a polynucleotide encoding an endoge Lombardi et al., 1990, J. Arachnol., 18(3):297-306. nous spider silk protein or variant thereof. The sequenc Abstract, accession No. 008445422 of DIALOG File ing of a spider silk protein is made possible by a method 351 of JP 2240165, published Sep. 25, 1990. for solubilizing a spider silk protein. Abstract, accession No. 008287285 of DIALOG File 351 of JP 2113066, published Apr. 25, 1990. 3 Claims, 1 Drawing Sheet U.S. Patent Sep. 14, 1993 5,245,012 FIG.1 U A-AVA - I - I - AUU-U-OA- UV-to-VA FIG.2 FIG.4 5,245,012 1. 2 the seven types of silks, only two have been investigated METHOD TO ACHIEVE SOLUBILIZATION OF in any detail, and no sequence data were obtained. SPIDER SLK PROTEIN'S In addition to the problem of solubility, multiple forms of spider silks are produced simultaneously by STATEMENT OF GOVERMENTAL INTEREST 5 any given spider. The resulting mixture is much less The invention described herein may be manufac useful than a single isolated silk because the different tured, used and licensed by or for the Government for spider-silk proteins have different properties and, due to Governmental purposes without the payment to us of solubilization problems, are not easily separated by any royalty thereon. methods based on their physical characteristics. This application is a division of application Ser. No. 10 Accordingly, at least three major problems have 511,114 filed Apr. 19, 1990, now abandoned. inhibited consideration of the feasibility of producing spider silk fibers with desirable characteristics, in com BACKGROUND OF THE INVENTION mercially useful quantities, for use as components of The present invention relates to producing a spider textile, composite and ballistic materials. First, an effec silk protein, protein fragment or variant, in commer 15 tive means for solubilizing silk protein has been unavail cially useful amounts, by means of a recombinant host able heretofore, making amino-acid sequencing of the organism. protein virtually impossible. As a corollary, it has not There is considerable interest currently in making been possible to identify, isolate and clone a spider silk high-strength, light and versatile fibers. Most high encoding DNA into a suitable expression system. Nor strength fibers used today, such as Nylon TM and the 20 has it been feasible to produce a spider silk protein in synthetic ballistic fiber Kevlar TM, have a high density, quantities much greater than can be obtained naturally are expensive and are limited in their range of use. If it from spiders. Consequently, there has been a need but were available in commercially useful amounts, a natu no means for providing commercially useful quantities rally occurring protein could provide an alternative of spider silk protein in a form displaying homogeneous fiber with enhanced properties. 25 mechanoelastic properties. Spider silks have been demonstrated to have several desirable characteristics. For example, spider silk could SUMMARY OF THE INVENTION be used as a light-weight, high-strength fiber for various It is therefore an object of the present invention to textile and ballistics applications, as well as for compos provide commercially useful quantities of a homogene ite materials. Spider silks represent a very diverse group 30 ous spider silk protein. In this regard, a "homogeneous" of fibers, particularly with respect to their mechanoelas silk-protein composition is one that, while possibly con tic properties, which in turn are largely a function of taining more than one type of fiber, possesses uniform fiber composition and molecular conformation. The mechanostructural properties such as tensile strength, spider silks range from those displaying a tensile 35 energy required to break a fiber, elasticity, ballistic limit strength greater than steel (7.8 vs 3.4 G/denier) and and modulus. those with an elasticity greater than wool (46% vs. 43% Another object of the present invention is to provide extension to break) to others characterized by energy a method for producing a recombinant spider silk pro to-break limits that are greater than Kevlar TM (1 x 105 tein in recoverable amounts. vs 3x104JKG-1). Yet another object of the present invention is to pro Considerable difficulty has been encountered in at vide a method for solubilizing a spider silk protein. tempting to solubilize and purify natural spider silk In accomplishing the foregoing objectives, there has while retaining the molecular-weight integrity of the been provided, in accordance with one aspect of the fiber. Another disadvantage of spider silk protein is that present invention, an isolated spider silk protein com only small amounts are available from cultivated spi 45 prising an amorphous domain or subunit and a crystal ders, making commercially useful quantities of silk pro line domain or subunit. In this context, a domain refers tein unattainable at a reasonable cost. to a portion of an endogenous protein that provides The term "fibroin' is often used for the silk fibers particular mechanostructural properties to the protein secreted by some insects and arachnids. See, e.g., Lucas and a subunit refers to a given amino-acid sequence that et al., Ady. Protein Chem. 13:107-242 (1958). Studies of 50 is repeated in the protein. the chemistry of these fibroins have been reported, for In a preferred embodiment, a crystalline domain or example, by Work and Young, J. Arachnol. 15:65-80 subunit comprises the amino-acid sequence Ala-Gly (1987). Nevertheless, only limited data are available on Ala-Gly-Ala-Gly-Ala-Gly-Tyr-Gly-Ala-Gly-Ala-Gly the composition of silk fiber from spiders, including Ala-Gly-Ala-Gly-Ala-Gly-Tyr-Gly-Ala-Ala-Ser -Gly those of the genus Nephila. For example, partial amino 55 Ala-Gly-Ala-Gly-Ala-Gly-Glu-Gly-Ala-Gly-Glu-Gly acid constituency has been reported for silks of N. Gly-Ala-Gly-Glu-Gly-Glu-Gly-Ala-Gly-Glu-Gly senegalensis and N. madagascar lensis, Lucas et al., J. -Ala-Gly-Tyr-Gly-Tyr. In another preferred embodi Mol. Biol. 2:339-49 (1960); and N. clavipes, Tillinghast & ment, an amorphous domain or subunit comprises the Christensen, J. Arachnol. 10: 69-74 (1984). While these amino-acid sequence Ala-Gly-Ala-Gly-Tyr-Gly-Ala investigations suggest that the different Nephila silks 60 -Ala-Ser-Arg-Leu-Thr-Cys-Gly-Thr-Pro-Gly-Ser-Gly vary in composition and properties, there is insufficient -Gln-Gly-Phe-Asp-Tyr-Arg-Ile-Arg-Arg-Glu-Gly-Tyr information to make a definitive correlation between -Gly-Gly-Leu-Gly-Arg-Arg-Glu-Gly-Tyr-Gly-Gly chemical composition and structural properties.