United States Patent (19) 11 Patent Number: 5,245,012 Lombari Et Al

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. Leu. In alternative preferred embodiment an amor The silk fibers of Nephila spiders are synthesized by phous domain or subunit comprises the amino-acid se specialized glands situated in the abdominal cavity. 65 quence Arg-Arg-Glu-Gly-Tyr-Gly-Gly-Leu-Gly. In a Andersen has reported on the amino acid compositions further preferred embodiment, a spider silk protein or for the seven silks obtained from one animal. See An variant has an N-terminal sequence of Ile-Ser-His-Val dersen, Como. Biochem. Physiol. 35:705-711 (1970). Of Pro-Thr-His-Glu-Asp-Glu-Ser-Ala-Ala -Val-Gly-Ala 5,245,012 3 4. Gly-Ala-Gly-Ala-Gly-Ala-Ala-Ala-Gly -Ser-Gly-Ala FIG. 2 depicts a an exemplary spider silk protein or Gly-Ala. variant amorphous domain or subunit and correspond In accordance with another aspect of the present ing anti-sense cDNA sequence. invention a homogeneous spider silk protein composi FIG. 3 depicts an alternative dragline silk protein tion is provided. Such a composition can comprise an amorphous domain or subunit and corresponding anti isolated spider silk protein or spider silk variant having sense cDNA sequence. at least one crystalline domain and at least one amor FIG. 4 depicts a portion of a dragline silk protein or phous domain. Another aspect of the present invention variant crystalline domain or subunit and corresponding to provide an isolated spider silk protein or variant anti-sense cDNA sequence. wherein the ratio of the crystalline domain to the amor O In these drawings, an anti-sense cDNA sequence phous domain is greater than 1, such that the tensile corresponds to the encoding mRNA sequence except stength of the resulting spider silk is increased. Alterna that "T" in a cDNA sequence designates a thymine tively, the ratio of the crystalline domain to the amor base, while, in the corresponding RNA sequence, T is phous domain is less than 1, such that the elasticity of replaced with “U” to designate a uracil base. the resulting spider silk is increased. Another aspect of DETAILED DESCRIPTION OF THE the present invention is to provide an isolated spider silk PREFERRED EMBOOMENTS protein or variant that is in substantially pure form. Pursuant to the present invention, spider silk proteins Still another aspect of the present invention is to can be solubilized in a manner that permits their se provide a polynucleotide encoding a spider silk protein 20 quencing and purification. Moreover, the sequence or variant, a vector comprising such a polynucleotide, information thereby obtained enables the cloning of such that the vector can be selected form the group spider silk-encoding DNA and the heterologous expres consisting of a viral vector, a phage vector, a cosmid, sion of spider silk proteins in commercially useful quan and a plant vector. In addition the present invention tities. Polypeptides of the present invention therefore provides a host cell comprising a polynucleotide as 25 include recombinant spider silk proteins, as well as frag described above, selected from the group consisting of ments and variants thereof, as defined below, that are a bacterial cell, an insect cell, a yeast cell, a mammalian commercially useful as components of textile, compos cell, and a plant cell. ite and ballistic materials. By the same token, the pres An additional aspect of the present invention is to ent invention includes polynucleotides that code for provide a method for producing a recombinant spider 30 such spider silk proteins and variants. silk protein comprising the steps of providing a host cell More specifically, a method within the present inven comprising an isolated polynucleotide encoding a spi tion for producing a recombinant spider silk protein or der silk protein or variant, culturing the host cell such variant involves the steps of providing a host cell hav that said spider silk protein or variant is expressed by ing a heterologous polynucleotide which encodes a said host cell in recoverable amounts; and recovering 35 spider silk protein, culturing the host cell under condi the spider silk protein or variant. tions such that the protein can be produced by the host Another aspect of the present invention is to provide cell in recoverable amounts, and recovering the protein a method for solubilizing a spider silk protein or variant, in a substantially pure form that is suitable for commer comprising the steps of providing a sample comprising cial applications. at least one spider silk protein or variant, contacting the 40 A "recoverable' amount in this regard means that an said sample with a solution consisting essentially of isolated amount of a spider silk protein can be detected propionate and hydrochloric acid in a 50%-50% vol by a methodology less sensitive than radiolabeling, such ume per volume (v/v) ratio, and solubilizing the spider as an immunoassay, and can be subjected to further silk protein or variant in the solution to obtain a solubi manipulations involving transfer of the protein per se 45 into solution. Preferably, a recoverable amount of a lized spider silk protein, such that the solubilized spider spider silk protein or variant should be an amount such silk protein is susceptible to amino-acid sequencing. that transferring the protein into solution yields a con Other objects, features and advantages of the present centration of at least 50 nM, preferably at least 50 uM. invention will become apparent to those skilled in the According to the present invention, spider silk prote art from the following detailed description. It should be 50 ins can be solubilized without disrupting protein struc understood, however, that the detailed description and ture to the extent that the molecular-weight integrity of specific examples, while indicating preferred embodi the protein is compromised. Solubilization to this end ments of the present invention, are given by way of involves the use of concentrated hydrochloric acid illustration and not limitation. Many changes and modi (HCl) of at least 6N concentration, optionally in con fications within the scope of the present invention may 55 junction propionic acid. In one embodiment, soluene be made without departing from the spirit thereof, and can be used as a less efficient solvent, but which is capa the invention includes all such modifications. Unless ble of partially dissolving a spider silk protein or vari otherwise indicated, the respective contents of the doc ant. In a preferred embodiment, the spider silk protein is uments cited below are hereby incorporated by refer contacted with constantly boiling mixture of 6N HC eCe. and 50% propionic acid (50:50, v/v). An acid mixture of this constituency dissolves spider silk protein and pro BRIEF DESCRIPTION OF THE DRAWINGS vides a clear, nonviscous solution which is suitable for The present invention may be more readily under use in determining amino acid sequence by known stood by referring to the accompanying drawings by methods, such as Edman degradation or hydrolysis which 65 HPLC. FIG. 1 depicts an amino-acid sequence and corre Spider silk proteins and variants of the present inven sponding anti-sense cDNA sequence of the N-terminus tion have commercially useful properties, suitable for of a exemplary spider silk protein. textile, composite and ballistic materials, including de 5,245,012 5 6 sirable tensile strength, elasticity, ballistic limit, and tural properties that are like those of the endogenous modulus. The properties of a spider silk protein or vari protein. ant of the present invention is determined by the rela Spider silk muteins can be produced, in accordance tive ratio of the amorphous and crystalline domains or with the present invention, by conventional site subunits. Modification of the relative and total amounts directed or cassette mutagenesis, two avenues for rou of these amorphous and crystalline domains or subunits tinely identifying residues of a spider silk protein which in a recombinant spider silk protein of the present inven can be modified without adversely affecting particular tion provide improved commercially useful properties, mechanoelastic properties. See Ausubel et al., CUR as described above. Examples of how modifications in RENT PROTOCOLS IN MOLECULAR BOLOGY this relative ratio will affect these properties include O (John Wiley & Sons 1987, 1990) (hereafter "Ausubel') increased tensile strength by increasing the crystalline at S8. Oligonucleotide-directed mutagenesis, compris to amorphous domain ratio, increased elasticity by ei ing i) synthesis of an oligonucleotide with a sequence ther increasing the amorphous to crystalline ratio or that contains the desired nucleotide substitution (muta decreasing the occurrence of Ala-Ala dipeptides in the tion), (ii) hybridizing the oligonucleotide to a template crystalline domains. These modifications of the struc 15 comprising a structural sequence coding for a spider silk ture of an endogenous spider silk protein can be accom protein or variant and (iii) using T4DNA polymerase to plished by conventional procedures such as site extend the oligonucleotide as a primer, is preferred directed or cassette mutagenesis of isolated polynucleo because it is readily applied in determining the effect(s) tides that encode functional portions of a spider silk of particular changes to a spider silk protein structural protein or variant. sequence. Its relative expense may militate in favor of A recombinant spider silk protein of the present in an alternative, known direct-mutagenesis method. vention can be obtained in recoverable amounts in a A spider silk variant that correspond to a portion of a form such that the spider silk protein preparation mi spider silk protein would be a polypeptide containing grates as a single band on a silver stained and commassie the amino-acid sequence that corresponds to at least one blue stained SDS-PAGE gel ("substantially pure 25 repeating structural unit of an amorphous or crystalline form'). In terms of relative purity, a preferred form of domain of a spider silk protein, absent the other struc a recombinant spider silk protein is one that provides a tural portions. In this context, the ratio of amorphous single peak in a conventional high-performance liquid and crystalline domains or subunits could be increased chromatography column. or decreased to provide a recombinant spider silk vari Based on the nucleotide sequences that encode poly 30 ant that had varied mechanostuctural properties, as peptides, e.g., as set out in FIGS. 1-4, and on knowl desired for a particular textile, composite or ballistic edge regarding newly characterized amorphous and material. For example, decreasing the ratio of amor crystalline domains of spider silk proteins, polypeptide phous subunits to crystalline subunits can increase the molecules can also be produced which represent varia tensile strength of a spider silk variant according to the tions of the naturally occurring molecule. The charac 35 present invention. terization of these crystalline and amorphous domains Examples of crystalline and amorphous domains are or subunits is carried out by analysis of the discovered those of a spider dragline silk from Nephila clavipes, amino acid sequences of a spider silk protein. The crys wherein a crystalline domain or subunit can comprise talline domains or subunits of a spider silk protein or the amino-acid sequence Ala-Gly-Ala-Gly -Ala-Gly variant of the present invention are characterized by Ala-Gly-Tyr-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly comparison of the discovered spider silk protein amino Ala-Gly-Tyr-Gly-Ala-Ala-Ser-Gly-Ala-Ala -Gly-Glu acid sequence with known crystalline domains of Bon Gly-Glu-Gly-Ala-Gly-Glu-Gly-Ala-Gly-Tyr-Gly-Tyr. byx mori. See, e.g., Iizuka Biorheology 3:551-552 (1965). An amorphous domain or subunit can similarly com Alternatively, the amorphous domains or subunits are prise the amino-acid sequence Ala-Gly-Ala-Gly-Tyr characterized by finding repeated, non-crystallinese 45 Gly-Ala-Ala-Ser-Arg-Ile-Thr-Cys-Gly-Thr -Pro-Gly quences within the discovered spider silk amino acid Ser-Gly-Gln-Gly-Phe-Asp-Tyr-Arg-Ile-Arg-Arg-Glu sequences. Gly-Tyr-Gly-Gly-Leu-Gly-Arg-Arg-Glu-Gly-Tyr These polypeptide molecules that contain variations Gly-Gly-Leu. An alternative amorphous domain or of the ratio and amounts of endogenously occuring subunit can also comprise the amino-acid sequence Arg amorphous and crystalline domains are referred to here 50 Arg-Glu-Gly-Tyr-Gly-Gly-Leu-Gly. A recombinant generically as "spider silk variants' and include, for spider silk protein or variant can have an N-terminal example, spider silk muteins and molecules that corre sequence of Ile-Ser-His-Val-Pro-Thr-His -Glu-Asp spond to portions of a spider silk protein. The key to Glu-Ser-Ala-Ala-Val-Gly-Ala-Gly-Ala-Gly --Ala-Gly diversifying the silks is in altering the genetic makeup of Ala-Ala-Ala-Gly-Ser-Gly-Ala-Gly-Ala. the silk-encoding polynucleotide to tailor the physico 55 Other spider silk variants within the present invention chemical makeup of the expressed spider silk polypep can be fragments of the cloned and expressed molecule tide for various fiber applications. that retain at least one commercially useful mechano In this regard, a "spider silk mutein' is a polypeptide elastic property of a spider silk protein, and that are that retains the basic structural attribute of spider silk homologous to a spider silk protein. Such fragments s-namely, at least one repeated amino-acid sequence could be produced by known de novo-synthesis tech representing an amorphous and/or a crystalline subunit niques and by fragmentation of the spider silk protein of an endogenous silk protein-and a commercially molecule itself, as well as by producing a genetically useful mechanoelastic property of a spider silk protein. engineered vector/host cell system that expresses a A spider silk mutein can also be homologous to an en spider silk protein fragment encoded by a heterologous dogenous spider silk protein. "Homology" in this con polynucleotide used to transform the host. text connotes a degree of similarity in amino acid se To be used in recombinant expression of a spider silk quence, relative to an endogenous spider silk, such that protein or a spider silk protein variant, a polynucleotide the mutein in question displays typifying mechanostruc molecule encoding a spider silk protein or a spider silk 5,245,012 7 8 protein variant would preferably comprise a nucleotide nucleotide encoding, e.g., a portion of the N-terminus sequence, corresponding to a desired amino-acid se or known domains of a spider silk protein or variant of quence, that is optimized for the host cell of choice (see the present invention, e.g., as a crystalline domain com below) in terms of codon usage, initiation of translation prising a portion of the amino acid sequence depicted in and expression of recoverable amounts of a commer FIG. 4, such as GGC-GAA-GGC-GCT-GGC-GAG cially useful spider silk protein or a spider silk protein GGC-GGT-GCT; an amorphous domain comprising a variant. Also, the vector selected for transforming a portion of an amino acid sequence depicted in FIG. 1, chosen host organism with such a polynucleotide mole such as GGA-TAT-GGC-GGT-CTA-GGA or an al cule should allow for efficient maintenance and tran ternative amorphous domain comprising an amino-acid scription of the sequence encoding the polypeptide. O sequence Arg-Arg-Glu-Gly-Tyr-Gly-Gly-Leu-Gly. In Vectors can be used for cloning and expression of a a further preferred embodiment, the spider silk protein polynucleotide according to the present invention in a has an N-terminal sequence of Ile-Ser-His-Val-Pro-Thr host cell. Such vectors can be derived, for example, His-Glu -Asp-Glu-Ser-Ala-Ala-Val-Gly-Ala-Gly-Ala from a bacteria, a virus, a filamentous phage (such as a Gly-Ala-Gly-Ala-Ala-Ala-Gly-Ser-Gly-Ala-Gly-Ala. M-13 derived phage), a cosmid, a yeast or a plant. Vec 15 See Ausubel at S6. tors will preferably include a replicator, a selectable Alternatively, other portions including or adjacent to marker, and a cloning site. See Ausubel at S 1.51. Proto an endogenous coding sequence of a spider silk protein cols for obtaining and using such vectors are known to or variant according to the present invention can be those in the art. Ausubel at SS 1.5-1.15, 9.1-9.6 and used, when isolated using a probe as a template for 13.4-13.11. 20 generating other probes useful for isolating a spider silk Examples of bacteria-derived vectors include plasmid protein or variant-encoding polynucleotide according vectors such as pBR322, puC19, pSP64, pluR278 and to the present invention, e.g., based on the N-terminal, pORF1. Ausubel at S 1.5. Illustrative of suitable viral crystalline or amorphous domain sequences described vectors are those derived from phage, vaccinia, re above. Such a probe can be used by known procedures trovirus, baculovirus, or a bovine papilloma virus. Ex 25 for screening a genomic or cDNA library as described amples of phage vectors include A, AEMBL3, A2001, above, or as a basis for synthesizing PCR probes for Agt 10, Agtl 1, Charon 4a, Charon 40, and AZAP/R. See amplifying a cDNA generated from an isolated RNA id. at SS 1.10-1.12. pKB3 and pSC11 are exemplary of encoding a spider silk protein or variant according to vaccinia vectors (see, e.g., Chakrabarti et al., Molec. the present invention. For example, transformants can Cell. Biol. 5:3401-9 (1985) and Mackett et al J. Virol. 30 be selected for expression by a host cell of a spider silk 49:857-864 (1984). Suitable retroviral vectors can also protein or variant, by use of selection media appropriate be used. See Hollis et al., Nature 296:321-325 (1982). to the vector used, RNA analysis or by the use of anti Among illustrative baculovirus vectors are pacRP23, bodies specific for a spider silk protein according to the described in Matsuura, J. Gen. Virol. 68:1233-50 (1987) present invention. See, e.g., Ausubel at SS9.5.2 (select and in Possee & Howard, Nucl. Acid Res. 5:10233-48 35 able markers), S9.8 (RNA analysis), SS.10.6-8 (detection (1987). An example of a filamentous phage vector is an of proteins), SS 1.1-1.2 (immunoassays) and SS 11.3-16 M13-derived vector like M13mp18, M13mpl9/pUC19, (preparation and use of monoclonal, polyclonal and M13mpl8/pUC18, M13mplo/pUC13, antipeptide antibodies for protein detection). M13mp10/pUC12 and M13mp7/pUC7. See Ausubel at Such a cDNA can then be cloned into a suitable Sl. 14. Examples of a yeast vector include Yip5, YRp7, expression vector and used to transform a host cell, as YEp24, 2 um plasmid, YCp50, pYAC3 and Tri described below. See Ausubel at S15.4. Suitable host choderma reesei. Ausubel, SS13.4-13.6. Alternatively, cells in this context include prokaryotic cells (bacterial bovine papilloma-derived vectors can be used. See, e.g., or blue-green algal) and eukaryotic cells such as yeast, Dinaio et al Proc. Nat'l Acad. Sci., USA 79:4030-4034 insect, mammalian and human cells. Suitable host cells (1982) and Sekiguchi et al Gene 21:267-272 (1983). 45 of the present invention can preferably include microor When most or all of a relatively long polynucleotide ganisms, e.g., of the genera Aeromonas, Aspergillus, (for example, > 10kb) encoding a spider silk protein is Bacillis, Escherichia, Kluyveromyces, Pichia, Rhodo expressed according to the present invention, vectors coccus, Saccharomyces and Streptomyces. Illustrative are preferably selected that express such long polynu of microorganisms used as host cells according to the cleotides efficiently. For example, in the case of a poly 50 present invention include, as bacterial cells, E. coli and nucleotide encoding a dragline silk protein of N. cla Bacillus, and as yeast cells, Saccharomyces cerevisiae vipes, the golden orb-weaving spider (see below), the strains X2181-1B, with genotype a trp1 gall adel his2 polynucleotide is about 12 kb long. Suitable vectors for (available from the Yeast Genetic Stock Center, Berke expressing such a polynucleotide include those that ley, Calif.); strain ATCC 52683, with genotype a his2 efficiently express a polynucleotide that is greater than 55 adel trpl met14 ura 3 (aka strain "J17,' available from 11 kb, e.g., phage vectors such as EMBL3 or gtli or the American Type Culture Collection, Rockville, yeast vectors such as 2 um plasmid or pYACC3. See Md.); and strain ATCC 46183, with genotype a his1 Ausubel at SS 1.10 and 13.4. trp1 (aka "strain IL166-5B,' also available from the According to the present invention, DNA that en American Type Culture Collection). codes a spider silk protein or variant can be isolated and For introducing a polynucleotide of the present in expressed in a recombinant microbe or other organism, vention into a bacterial cell, known procedures can be by known procedures, to produce the desired polypep used according to the present invention such as by tide in commercially useful amounts. Such a DNA can transfection, e.g., using calcium phosphate precipita be isolated by screening nucleic acid libraries generated tion, electroporation, DEAE dextran, pelletizing with a from microorganisms expressing a spider silk protein or 65 DNA gun or using a recombinant phage virus. See variant according to the present invention. See Ausubel Ausubel, at S1.8. Other known procedures can also be at SS5 and 6. These libraries can be screened using oli employed to obtain a recombinant host cell that ex gonucleotide probes that are complementary to a poly presses a heterologous spider silk protein according to 5,245,012 10 the present invention, as will be apparent to those line) that possesses the highest strength. Three morpho skilled in the art. logical regions distinguish the major ampullate gland: For introducing a polynucleotide of the present in the tail, the sac and the duct. The tail is the site of about vention into a yeast cell, the most commonly used pro 90% of the major ampullate gland's protein synthesis tocol, the lithium acetate method, exploits the fact that activity; the ampulla is a storage site for soluble dragline alkalications make yeast cell membrane permeable to silk; and the duct appears to be involved with secretion DNA; in addition, uptake of foreign DNA is promoted and ordering of silk. Bell & Peakall, J. Cell Biol. by the presence in the medium of a high-molecular 42:285-95 (1969). weight molecule, polyethylene glycol. An alternative Major ampullate glands of N. clavipes, as in other method, spheroplast transformation, can be used but is 10 spiders, can be dissected and messenger RNA (mRNA) more time-consuming than the lithium acetate proce from the gland isolated and purified, in accordance with dure, though it results in a higher efficiency of transfor the present invention, by oligo d(T) cellulose chroma mation per input DNA. tography. Dragline silk cDNA can then be constructed Another type of suitable expression system of the by reverse-transcribing the gland mRNA. present invention entails the use of a mammalian host 15 In order to clone and express commercially useful cell transformed with a polynucleotide within the pres amounts of a spider silk protein, such as Nephila drag ent invention. Suitable vectors can be used that express line silk, a portion of the amino-acid sequence of the a spider silk protein or variant efficiently in mammalian natural protein can be determined, e.g., by Edman deg host cells such that the protein is expressed in commer radation, and synthetic oligonucleotide probes can be cially useful quantities. Illustrative of suitable mammali 20 constructed based on this sequence information, taking an-cellular hosts which can be used for this purpose are into account the redundancy of the codons encoding Chinese ovary (CHO) cells as described by Urlaub & such a protein. mRNA can be isolated by known proce Chasin, Proc. Nat'l Acad. Sci. USA 77:4216 (1980) and dures (See, e.g., Id., at S4) from the major ampulate baby hamster kidney (BHK) cells, exemplified by a cell gland which produce a silk protein. The mRNA is then line deposited under accession number ATCC CCL 10 25 reversed transcribed to construct a cDNA library, fol and another line ATCC CCL 70. lowed by screening with the above-mentioned probes For both yeast and mammalian expression systems, (see, e.g., Ausubel, at SS5 and 6, respectively). there are conventional transformation and screening The synthesized cDNA can be cloned into an expres protocols which can be employed pursuant to the pres sion vector and the lambda gt11 Sfi-Not vector (avail ent invention. Standard methodology in this regard is 30 able from Promega Biotech) can be employed. Lambda detailed in Ausubel, supra, at SS9 and 13. gtll Sfi-Not DNA is thus used as a vector for orienta For baculoviral expression systems, conventional tion-specific cDNA cloning, allowing the expression of transformation procedures with pACRP-derived vec cloned inserts as polypeptides fused with g-galactosi tors are used to transform suitable host cells including dase. Directional cloning can be achieved by using a those of, e.g., Spodoptera (such as sf cells), Tricho 35 unique oligodeoxynucleotide primer-adapter containing plusia, and heliothis. See Luckow & Summers, Biotech the recognition site for Not upstream from an oli nology 6:47-55 (1988); Miller, Ann. Rev. Microbiol. go(dT) sequence to prime first strand synthesis. After 42; 177-199 (1988); Maeda, Ann. Rev. of Microbiol. second strand synthesis and ligation of either EcoRI 34:351-72 (1989). For vaccinia viral expression systems, linkers or adapters, the double stranded molecules can see Chakrabarti et al., Molec. Cell. Biol. 5:3401-9 (1985) be digested with NotI (and EcoRI for linkers). Follow and Mackett et al., J. Virol. 49:857-864 (1984). While the ing removal of excess linker/adaptor fragments, the foregoing represents preferred methods and materials spider silk cDNA is ready to ligate into the provided for expressing spider silk proteins according to the pres EcoRI-Not vector arms. ent invention, it will be apparent to those of skill in the Recombinant phage can then be recognized by their art that many alternative methods are suitable for ex 45 ability to form colorless plaques when plated on lac pressing such a protein according to the present inven hosts (E. coli Y1089(r-) and Y1090(r)) in the presence of tion. X-GAL (5-bromo-4-chloro-3-indolyl-g-galactopyrano Introduction of a polynucleotide of the present inven side). By this method, the spider silk cDNA molecules tion into mammalian cells to produce a recombinant cell can be cloned in the same orientation relative to the which expresses a spider silk protein or variant can be 50 lacz gene, which can effectively double the likelihood accomplished according to conventional procedures, of in-frame expression of cloned silk genes as fusion such as by calcium phosphate or DEAE-dextran trans proteins. This strategy can increase the possibility of fection. See, e.g., Ausubel, supra, at S9. Expression of successful isolation of specific cDNA clones using nu such recombinant cells of the present invention pro cleic acid or antibody probes. vides recoverable amounts of a spider silk protein or 55 Dragline silk clones can be further isolated by using variant. nucleic acid probes constructed specifically from drag Host cells comprising a polynucleotide which en line silk protein sequences. Once identified, these clones codes a spider silk protein or variant of the present can be repurified to ensure purification of only the drag invention can be grown under conditions that provide line silk cDNA. Positive clones can be sequenced and expression of a desired polypeptide in recoverable or the overlapping sequences can be used to determine the commercially useful amounts. See id., SS 1 and 13. full length nucleic acid sequence that encodes a dragline An example of a silk spider protein suitable for solubl silk protein of Nephila. See Ausubel at SS 6 and 7. The ization according to the present invention is the silk of full length cDNA can then be reconstructed from the the golden orb-weaving spider, N. clavipes, a large spi cloned fragments. Id. at $3.16. der found in the tropical and subtropical areas of the This cDNA can be expressed in E. coli or other suit western hemisphere. Moore, Am. Mid Natur. 98.95-108 able host organism, and the presence of the resultant (1977). This species produces five to seven different silk silk/g-GAL fusion protein can be ascertained, for ex proteins, but it is the major ampullate gland silk (drag ample, by immunoscreening, thereby to identify recom 5,245,012 11 12 binants which produce the protein. Selected clones can then be cultured and tested, for example, by western TABLE 1-continued blotting for the presence of the protein. The protein can Solubility of Nephila clavipes be purified in a conventional manner, either from the dragline silk in various solvent systems. host medium or from a preparation of lyzed host cells, Solvent Solubility at Room Temperature for example, by using an immunoaffinity adsorbent col pionic Acid 1. Totally insoluble umn. By this approach, dragline spider silk protein from 2. Partially soluble, some particulates the species Nephila clavipes was found to have a molecu 3. Partially soluble, no particulates, viscous suspension lar weight of about 350,000 daltons and is encoded by a 4. Totally soluble, no particulates, clear, non-viscous polynucleotide of about 12,000 daltons. 10 The present invention is further described by refer Of the solubilizing agents studied, only hydro ence to the following, illustrative examples. In these chloric/propionic acid (50:50, viv) dissolved N. clavipes examples, specimens of the following arachnid species dragline silk at room temperature with only slight agita were used: N. clavipes Nephilinae, supplied by Angela tion (Table 1). Hydrochloric acid below 6N and used 15 alone failed to completely dissolve the silk even at ele Choate (USDA, Gainesville, Fla.); Argiope aurantia vated temperatures (data not shown). Some quaternary Lucas and Neoscona domiciliorum Hentz, supplied by ammonium compounds used as commercial tissue solu Mark Stowe, (University of Florida, Gainesville). Live bilizers proved to be efficient solvents, but the solvent specimens were housed in individual cages and fed a could not be easily removed from the solution. High diet of german cockroaches, Blatella germanica (Blattel 20 concentrations of base also dissolved silk samples, al lidae). Some specimens were frozen in liquid nitrogen though they were not used because the elevated tem and stored at -70° C. for subsequent nucleic acid ex peratures needed for solubilization may begin random tractions. hydrolysis of the silk backbone prior to amino acid EXAMPLE hydrolysis. Any amino acids hydrolyzed prior to the 25 150° C. hydrolysis reaction may then become com Silk Collection pletely degraded at the hydrolysis step and subse Samples were collected from the aranaid species N. quently unaccounted for in the final analysis. clavipes Nephilinae, Arglope aurantia Lucas, and Neos Hydrochloric/propionic acid proved to be most suit cona domicilicorum Hentz. Controlled silking was per able. The silk thus dissolved retained its structural integ formed as described by Work and Emerson, supra 30 rity, having the same molecular-weight value as that (1982). Controlled silking was restricted to the spiders determined by polyacrylamide gel electrophoresis and which were large enough to be easily manipulated with high performance liquid chromatography. out damaging the spider. The silking procedure consis tently averaged 5 to 30 minutes and 5.0-10.0 milligrams EXAMPLE 3 (mg) of major ampulate silk gland was routinely ob 35 Silk Hydrolysis tained. The mature female was continuously observed Major ampullate gland silk samples (2.0 mg) were under 60X magnification to substantiate the glandular first dissolved in 2.0 ml of a hydrochloric/propionic source of silk. All reeled samples were examined using acid mixture at room temperature for 20 min with slight a light microscope (100X objective, 12.5X ocular; vortexing. Solubilized samples (100 ul at 1.0 g/ul ) 1250X total magnification) to ensure that there was no were vacuum dried in pyrolyzed vials and purged with contamination by minor ampullate gland fibers. argon gas. Hydrolysis was carried out by placing 200 ul of constant boiling 6N HCl in the bottom of an acid EXAMPLE 2 resistant reaction vessel along with 2 sodium sulfite Silk Solubilization 45 crystals. The vessel was again purged with argon gas, Silk samples (approximately 1.0-2.0mg) were placed sealed under vacuum and placed at 150 C. for 1 hour. in 13X 100mm sterile glass borosilicate test tubes. The Argon was used as a purging gas because of its purity solvents listed in Table 1 were added to a final concen and because it contributes fewer artifact peaks in the tration of 1.0 ug/ul and solubility determined visually at subsequent analysis. Sodium sulfite was discovered to room temperature. 50 be useful as an oxygen scavenger and aids in the recov ery of cysteine, serine, and threonine (Ted Tanhauser, TABLE 1 Cornell University, personal communication). Solubility of Nephila clavipes Multiple analyses were carried out on a Waters dragline silk in various solvent systems. HPLC Pico-Tag Amino Acid analysis system. The Solvent Solubility at Room Temperature 55 hydrolyzed samples were derivatized with phenyliso Water thiocyanate (PITC) and these samples reconstituted in NHC 400 ul of sample diluent. For each analysis a 50 ul injec tion volume was used. Amino acid standards were run with each sample. Ribonuclease A was run as an hydro lysis control. 1N KOH EXAMPLE 4 Chloroform Ethyl alcohol 95% Amino Acid Analysis 8M Urea The amino acid composition of the secretion of 50% Lithium Bromide % SOS (MaAS) from N. clavipes is shown in Tables 2 and 3. 5% Mercaptoethanol Glycine, alanine, glutamic acid/glutamine and arginine Soluene were the most abundant amino acids, comprising 74% Boiling 5N HC1/50% Pro of all amino acids present. Generally, the major ampul 5,245,012 13 14 late gland silk has been considered for use in the pro duction of dragline and frame threads of the web. The EXAMPLE 5 dragline has a high tensile strength (198 grams per de Cloning of a Spider Silk Protein nier, gpd) and it has a rupture elongation of 18%, deter A dragline silk of Nephila, as described in EXAM mined according to Zemlin, Technical Report 69-29- 5 PLES 1-4 above, was sequenced by Edman degrada CM, AD 684333 10760-5020 (1967). The composition tion, and synthetic oligonucleotide probes were con of the material from the large ampulate gland generally structed that corresponded to this sequence, as shown in agrees with the published analyses of dragline from N. FIG. 1, taking into account the redundancy of the co clavipes, see Zemlin, Ioc. cit., and Work & Young, J. dons encoding such a protein. A crystalline domain of Arachnol. 15:65-80 (1987), but some differences were O the spider silk protein was thus determined to have an observed. amino-acid sequence of Ala-Gly-Ala-Gly-Ala-Gly-Ala Table 3 shows the amounts of various amino acid side Gly-Tyr-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala chains in dragline silk of N. clavipes. Dragline silk is Gly-Tyr-Gly-Ala-Ala-Ser -Gly-Ala-Gly-Ala-Gly-Ala composed predominantly of the small side-chain amino Gly-Glu-Gly-Ala-Gly-Glu -Gly-Gly-Ala-Gly-Glu acids glycine, alanine and serine, which would allow 15 Gly-Glu-Gly-Ala-Gly-Glu-Gly-Ala-Gly-Tyr-Gly-Tyr. them to conform to the antiparallel beta-pleated sheet The probes were constructed by known methods. See model proposed by Pauling and Corey, Proc. Nat Acad. Ausubel at SS6.3-6.4. Sci. 39:253-256 (1953), for Bombyx mori. The conforma Glandular dissection was employed to obtain mRNA tional model applies only to the crystalline regions of B. encoding a dragline spider silk protein. Major ampulate mori, which makes up approximately 40% of the total glands were dissected out of living spiders through a 1.5 silk structure, as determined by x-ray diffraction analy cm longitudinal incision along the ventral abdomen. sis according to Lizuka, Biorheology 3:551-552 (1965). The glands were removed carefully to avoid degrada Limited x-ray diffraction data has been reported, how tion of the luminar contents. The glands were immedi ever, that indicate the degree of crystallinity in dragline ately transferred to a medium containing 0.10M sodium silk of certain araneid species. See Gosline et al., Nature 25 chloride and 0.015 M sodium citrate (SSC). Protease 309:551-552 (1984). inhibitors, phenylmethyl sulfonyl fluoride (PMSF) at a TABLE 2 final concentration of 6-10mg/ml (Methods in Enzymol Amino acid composition of the silk gland secretion of ogy 26:3-27 (1972)), and 20 units/ml of aprotonin Nephila clavipes. Results expressed as residues per 100 total. (Piperno et al., Proc. Natl. Acad. Sci. USA 74:1600-1604 Dragline spider l spider 2 spider 3 30 (1979)) were added to the dissection buffer to inhibit Trials 3 3. 3 proteases released by the gastric system of the spider. Asp/Asn (D/N) 2.5 2.4 2.6 Messenger RNA was isolated by known procedures Glu/Gln (E/Q) 9.1 9.0 9.2 Ser (S) 4.5 4.5 4.4 from the major ampullate gland which produces almost Gly (G) 37.0 37.3 36.9 35 exclusively dragline silk protein. See, e.g., Ausubel at His (H) O.S 0.4 0.4 S4. The mRNA was used to construct a cDNA library Arg (R) 7.6 7.6 7.7 Thr (T) 1.6 1.7 .6 and the above-mentioned probes were used to screen Ala (A) 21. 2.0 21.2 the library. Id. at SS5 and 6. Pro (P) 4.3 4.3 4.3 The synthesized cDNA was cloned into an expres Tyf (Y) 3.0 3.0 3.2 40 sion vector, the lambda gtll Sfi-Not vector (commer Wall (V) .8 1.8 1.7 Met (M) 0.3 0.3 0.2 cially available, e.g., from Promega Biotech, Promega Cys (C) 0.1 0. <0.1 Corporation, Madison, WI) was employed. Lambdagtill Ile (I) 10 10 1.0 Sfi-Not DNA is a vector designed for orientation spe Leu (L) 3.8 3.7 3.7 cific cDNA cloning which allows the expression of Phe (F) 0.7 0.7 0.6 cloned inserts as polypeptides fused with beta-galactosi Lys (K) 1.0 O O 45 dase. Directional cloning was achieved by using a unique oligodeoxynucleotide primer-adapter containing TABLE 3 the recognition site for Not upstream from an oli Amounts of various amino acid side chains in go(dT) sequence to prime first strand synthesis. After silk gland secretions of Nephila clavipes. 50 second strand synthesis and ligation of either EcoRI Results expressed as residues per 100 total. linkers or adapters, the double stranded molecules were Dragline silk spider 1 spider 2 spider 3 digested with NotI (and EcoRI for linkers). Following Trials 3 3 3 removal of excess linker/adaptor fragments, the spider small side chains 62.28 62.92 62.59 silk cDNA was ready to ligate into the EcoRI-Not polar side chains 29.8 29.6 30,22 55 vector arms provided. Recombinant phage are recog acidicamide side chains 1.67 1152 11.83 basic side chains 905 9.02 9.06 nized by their ability to form colorless plaques when cyclic innino side chain 4.3 4.34 4.28 plated on lac-hosts (E. coli Y1089(r) and Y1090(r-)) in aromatic side chain 3.62 3.57 3,88 the presence of X-Gal (5-bromo-4-chloro-3-indolyl-B- sulfur containing 0.47 0.46 0.22 aliphatic side chain 27.6 27.57 26.62 galactopyranoside). By this method, all of the spider silk hydroxyl side chain 6.16 6.20 cos 60 conA molecules were cloned in the same orientation smali side chains: gly-alaser relative to the lacz gene, which effectively doubled the polar residues: asp-thr-ser-glx -- tyr-lys --his--arg likelihood of in-frame expression of cloned silk genes as acidicamide residues: ask-gix fusion proteins. basic side chains: lys--his--arg cyclic imino side chain: pro Dragline silk clones were isolated by using nucleic aromatic side chain: phe-tyr 65 acid probes constructed specifically from dragline silk sulfur containing: met-cy's aliphatic side chain: ala -- valleu-like protein sequences. Once identified, these clones were hydroxyl side chain; ser-thr repurified 3x to ensure purification of only the dragline silk cDNA. Positive clones were sequenced by conven 5,245,012 15 16 tional procedures and the overlapping sequences used line and 10 amorphous domains, as depicted in FIGS. 4 to determine the full length nucleic acid sequence that and 2, respectively. encodes a dragline silk protein of Nephila. See, e.g., Ausubel at SS6 and 7. Next, the full length cDNA was EXAMPLE 8 reconstructed from the cloned fragments and was deter Cloning and Expression of a Spider Silk Mutein mined to have a length of 12 kb. Id. at $3.16. A mutein of a spider silk protein can be generated by EXAMPLE 6 modification of a cDNA encoding a spider silk protein as described in Example 5, above, by known site di Expression of a Spider Silk Protein rected or cassette mutagenesis techniques as described The cloned silk was expressed in E. coli via the vector O by, e.g., Ausubel at $8. Suitable portions of a cDNA can described in Example 5 above, and the resultant silk/g- be modified by site directed or cassette mutagenesis to Gal fusion protein was screened with a Protoblot in provide cDNA that encodes a dragline silk protein munoscreening system by screening phage plaques con mutein having a at least one of each of the amorphous taining the recombinant protein and by western blotting and crystalline domains depicted in FIGS. 2 and 4, of lambda lysogens after purification on a protosorb 5 respectively. When such a cDNA is expressed accord lacz immunoaffinity adsorbent column. The purifica ing to Example 6, above, a spider silk protein mutein tion on the Lacz immunoaffintiy column was alterna can be expressed that has a molecular weight of about tively used to elute the silk protein, cleaved from the 85,000 wherein the mutein comprises about 10 crystal lacz protein by treatment with cyanogen bromide, 20 line and 5 amorphous domains, as depicted in FIGS. 4 using increased salt concentration. The cyanogen bro and 2, respectively. Such a mutein would have higher mide and salt were then removed from the purified silk tensile strength than an endogenous spider silk protein protein by dialysis. because the ratio of crystalline domains to amorphous domains would be greater than 1. After full characterization of the recombinant protein Alternatively, a cDNA is expressed according to (MW =350,000), it was concluded that a recombinant 25 spider silk protein had been successfully cloned, ex Example 6, above, and a spider silk protein mutein can pressed, and isolated. Comparison of amino acid com be expressed that has a molecular weight of about position of the recombinant protein with the endoge 80,000 wherein the mutein comprises about 5 crystalline nous protein showed that while the amino acid compo and 10 amorphous domains, as depicted in FIGS. 4 and sition was not identical to either that of the spun spider 30 2, respectively. Such a mutein would have higher elas silk or that obtained from the major ampullae, the com ticity than an endogenous spider silk protein because position was similar enough to have the same me the ratio of crystalline domains to amorphous domains chanostructural and other physical characteristics of would be less than 1. the endogenous proteins. What is claimed is: 1. A method for solubilizing a spider silk protein or EXAMPLE 7 35 variant comprising the steps of: (A) providing a sample comprising at least one spider Cloning and Expression of a Spider Silk Protein Variant silk protein or variant; A variant of a spider silk protein can be generated by (B) contacting said sample with a solution consisting modification of a cDNA encoding a spider silk protein essentially of propionate and hydrochloric acid in a as described in Example 5, above, by known site di 50-50% volume per volume ratio; rected or cassette mutagenesis techniques according to, (C) solubilizing said spider silk protein or variant in e.g., Ausubel at S8. Suitable fragments of the cDNA can said solution to obtain a solubilized spider silk pro be modified by site directed or cassette mutagenesis to tein or variant. provide cDNA that encodes a dragline silk protein 2. A method according to claim 1, wherein said solu variant having a at least one of each of the amorphous 45 bilized spider silk protein or variant is susceptible to and crystalline domains depicted in FIGS. 2 and 4, amino-acid sequencing. respectively. When such a cDNA is expressed accord 3. A method according to claim 1, wherein said hy ing to Example 6, above, a spider silk protein variant drochloric acid is provided into said solution at a con can be expressed that has a molecular weight of about centration of at least 6 N. concentration. ll.0,000, wherein the variant comprises about 10 crystal 50 k k