WO1991016351A1 - Proteines de soie arachneennes recombinees obtenues par genie genetique - Google Patents

Proteines de soie arachneennes recombinees obtenues par genie genetique Download PDF

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WO1991016351A1
WO1991016351A1 PCT/US1991/002222 US9102222W WO9116351A1 WO 1991016351 A1 WO1991016351 A1 WO 1991016351A1 US 9102222 W US9102222 W US 9102222W WO 9116351 A1 WO9116351 A1 WO 9116351A1
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gly
ala
spider silk
silk protein
variant
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Stephen J. Lombardi
David L. Kaplan
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United States Department of the Army
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders

Definitions

  • the present invention relates to producing a spider silk protein, protein fragment or variant, in commercially useful amounts, by means of a recombinant host organism.
  • Spider silks have been demonstrated to have several desirable characteristics. For example, spider silk could be used as a light-weight, high- strength fiber for various textile and ballistics applications, as well as for composite materials. Spider silks represent a very diverse group of fibers, particularly with respect to their mechanoelastic properties, which in turn are largely a function of fiber composition and molecular conformation.
  • the spider silks range from those displaying a tensile strength greater than steel (7.8 vs 3.4 G/denier) and those with an elasticity greater than wool (46% vs 43% extension to break) to others characterized by energy-to-break limits that are greater than KevlarTM (1x10 s vs 3x10 JKG-1) .
  • spider silk protein is that only small amounts are available from cultivated spiders, making commercially useful quantities of silk protein unattainable at a reasonable cost.
  • fibroin is often used for the silk fibers secreted by some insects and arachnids. See, e.g., Lucas et al., Adv. Protein Chem. 13:107-242 (1958) . Studies of the chemistry of these fibroins have been reported, for example, by Work and Emerson, J. Arachnol. 15:65-80 (1987). Nevertheless, only limited data are available on the composition of silk fiber from spiders, including those of the genus Nephila . For example, partial amino-acid constituency has been reported for silks of N. senegalensis and N. madagascar lensi ⁇ , Lucas et al., J. Mol. Biol.
  • the silk fibers of Nephila spiders are synthesized by specialized glands situated in the abdominal cavity. Andersen has reported on the amino acid compositions for the seven silks obtained from one animal. See Andersen, Comp. Biochem. Physiol. 35:705-711 (1970). Of the seven types of silks, only two have been investigated in any detail, and no sequence data were obtained.
  • a "homogeneous" silk-protein composition is one that, while possibly containing more than one type of fiber, possesses uniform mechanostructural properties such as tensile strength, energy required to break a fiber, elasticity, ballistic limit and modulus.
  • Another object of the present invention is to provide a method for producing a recombinant spider silk protein in recoverable amounts.
  • Yet another object of the present invention is to provide a method for solubilizing a spider silk protein.
  • an isolated spider silk protein comprising an amorphous domain or subunit and a crystalline domain or subunit.
  • a domain refers to a portion of an endogenous protein that provides particular mechanostructural properties to the protein and a subunit refers to a given a ino-acid sequence that is repeated in the protein.
  • a crystalline domain or subunit comprises the amino-acid sequence Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Tyr-Gly-Ala-Gly- Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Tyr-Gly-Ala-Ala-SEQ-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Glu-Gly-Ala-Gly- Glu-Gly-Gly-Ala-Gly-Glu- Gly-Ala-Gly-Glu- Gly-Ala-Gly-Tyr-Gly-Tyr.
  • an amorphous domain or subunit comprises the amino-acid sequence Ala-Gly-Ala-Gly-Tyr-Gly- Ala-Ala-Ser-Arg-Ile-Thr-Cys-Gly-Thr-Pro-Gly-Ser- Gly-Gln-Gly-Phe-Asp-Tyr-Arg-Ile-Arg-Arg-Glu-Gly- Tyr-Gly-Gly-Leu-Gly-Arg-Arg-Glu-Gly-Tyr-Gly-Gly- Leu.
  • an amorphous domain or subunit comprises the amino- acid sequence Arg-Arg-Glu-Gly-Tyr-Gly-Gly-Leu-Gly.
  • a spider silk protein or variant has an N-terminal sequence of Ile-Ser-His-Val-Pro-Thr-His-Glu-Asp-Glu-Ser-Ala- Ala-Val-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Ala-Gly-Ser-Gly-Ala-Gly-Ala.
  • a homogeneous spider silk protein composition is provided.
  • Such a composition can comprise an isolated spider silk protein or spider silk variant having at least one crystalline domain and at least one amorphous domain.
  • Another aspect of the present invention to provide an isolated spider silk protein or variant wherein the ratio of the crystalline domain to the amorphous domain is greater than 1, such that the tensile stength of the resulting spider silk is increased.
  • the ratio of the crystalline domain to the amorphous domain is less than 1, such that the elasticity of the resulting spider silk is increased.
  • Another aspect of the present invention is to provide an isolated spider silk protein or variant that is in substantially pure form.
  • Still another aspect of the present invention is to provide a polynucleotide encoding a spider silk protein or variant, a vector comprising such a polynucleotide, such that the vector can be selected form the group consisting of a viral vector, a phage vector, a cosmid, and a plant vector.
  • the present invention provides a host cell comprising a polynucleotide as described above, selected from the group consisting of a bacterial cell, an insect cell, a yeast cell, a mammalian cell, and a plant cell.
  • An additional aspect of the present invention is to provide a method for producing a recombinant spider silk protein comprising the steps of providing a host cell comprising an isolated polynucleotide encoding a spider silk protein or variant, culturing the host cell such that said spider silk protein or variant is expressed by said host cell in recoverable amounts; and recovering the spider silk protein or variant.
  • Another aspect of the present invention is to provide a method for solubilizing a spider silk protein or variant, comprising the steps of providing a sample comprising at least one spider silk protein or variant, contacting the said sample with a solution consisting essentially of propionate and hydrochloric acid in a 50%-50% volume per volume (v/v) ratio, and solubilizing the spider silk protein or variant in the solution to obtain a solubilized spider silk protein, such that the solubilized spider silk protein is susceptible to amino-acid sequencing.
  • FIGURE 1 depicts an amino-acid sequence and corresponding anti-sense cDNA sequence of the N- terminus of a exemplary spider silk protein.
  • FIGURE 2 depicts a an exemplary spider silk protein or variant amorphous domain or subunit and corresponding anti-sense cDNA sequence.
  • FIGURE 3 depicts an alternative dragline silk protein amorphous domain or subunit and corresponding anti-sense cDNA sequence.
  • FIGURE 4 depicts a portion of a dragline silk protein or variant crystalline domain or subunit and corresponding anti-sense cDNA sequence.
  • an anti-sense cDNA sequence corresponds to the encoding mRNA sequence except that "T" in a cDNA sequence designates a thymine base, while, in the corresponding RNA sequence, T is replaced with "U” to designate a uracil base.
  • spider silk proteins can be solubilized in a manner that permits their sequencing and purification. Moreover, the sequence information thereby obtained enables the cloning of spider silk-encoding DNA and the heterologous expression of spider silk proteins in commercially useful quantities.
  • Polypeptides of the present invention therefore include recombinant spider silk proteins, as well as fragments and variants thereof, as defined below, that are commercially useful as components of textile, composite and ballistic materials.
  • the present invention includes polynucleotides that code for such spider silk proteins and variants.
  • a method within the present invention for producing a recombinant spider silk protein or variant involves the steps of providing a host cell having a heterologous polynucleotide which encodes a spider silk protein, culturing the host cell under conditions such that the protein can be produced by the host cell in recoverable amounts, and recovering the protein in a substantially pure form that is suitable for commercial applications.
  • a “recoverable” amount in this regard means that an isolated amount of a spider silk protein can be detected by a methodology less sensitive than radiolabeling, such as an immunoassay, and can be subjected to further manipulations involving transfer of the protein per se into solution.
  • a recoverable amount of a spider silk protein or variant should be an amount such that transferring the protein into solution yields a concentration of at least 50 nM, preferably at least 50 ⁇ M.
  • spider silk proteins can be solubilized without disrupting protein structure to the extent that the molecular- weight integrity of the protein is compromised. Solubilization to this end involves the use of concentrated hydrochloric acid (HCl) of at least 6N concentration, optionally in conjunction propionic acid.
  • HCl concentrated hydrochloric acid
  • soluene can be used as a less efficient solvent, but which is capable of partially dissolving a spider silk protein or variant.
  • the spider silk protein is contacted with constantly boiling mixture of 6N HCl and 50% propionic acid (50:50, v/v). An acid mixture of this constituency dissolves spider silk protein and provides a clear, nonviscous solution which is suitable for use in determining amino acid sequence by known methods, such as Edman degradation or hydrolysis-HPLC.
  • Spider silk proteins and variants of the present invention have commercially useful properties, suitable for textile, composite and ballistic materials, including desirable tensile strength, elasticity, ballistic limit, and modulus.
  • the properties of a spider silk protein or variant of the present invention is determined by the relative ratio of the amorphous and crystalline domains or subunits. Modification of the relative and total amounts of these amorphous and crystalline domains or subunits in a recombinant spider silk protein of the present invention provide improved commercially useful properties, as described above.
  • Examples of how modifications in this relative ratio will affect these properties include increased tensile strength by increasing the crystalline to amorphous domain ratio, increased elasticity by either increasing the amorphous to crystalline ratio or decreasing the occurrence of Ala-Ala dipeptides in the crystalline domains.
  • modifications of the structure of an endogenous spider silk protein can be accomplished by conventional procedures such as site-directed or cassette mutagenesis of isolated polynucleotides that encode functional portions of a spider silk protein or variant.
  • a recombinant spider silk protein of the present invention can be obtained in recoverable amounts in a form such that the spider silk protein preparation migrates as a single band on a silver stained and commassie blue stained SDS-PAGE gel ("substantially pure form”) .
  • a preferred form of a recombinant spider silk protein is one that provides a single peak in a conventional high-performance liquid chromatography column.
  • polypeptide molecules can also be produced which represent variations of the naturally occurring molecule.
  • the charaterization of these crystalline and amorphous domains or subunits is carried out by analysis of the discovered amino acid sequences of a spider silk protein.
  • the crystalline domains or subunits of a spider silk protein or variant of the present invention are charaterized by comparison of the discovered spider silk protein amino acid sequence with known crystalline domains of Bombyx mori . See, e.g., Iizuka Biorheology 3:551-552
  • the amorphous domains or subunits are characterized by finding repeated, non- crystalline sequences within the discovered spider silk amino acid sequences.
  • These polypeptide molecules that contain variations of the ratio and amounts of endogenously occuring amorphous and crystalline domains are referred to here generically as "spider silk variants" and include, for example, spider silk muteins and molecules that correspond to portions of a spider silk protein. The key to diversifying the silks is in altering the genetic makeup of the silk- encoding polynucleotide to tailor the physicochemical makeup of the expressed spider silk polypeptide for various fiber applications.
  • a "spider silk mutein” is a polypeptide that retains the basic structural attribute of spider silks — namely, at least one repeated amino-acid sequence representing an amorphous and/or a crystalline subunit of an endogenous silk protein — and a commercially useful mechanoelastic property of a spider silk protein.
  • a spider silk mutein can also be homologous to an endogenous spider silk protein.
  • "Homology” in this context connotes a degree of similarity in amino acid sequence, relative to an endogenous spider silk, such that the mutein in question displays typifying mechanostructural properties that are like those of the endogenous protein.
  • Spider silk muteins can be produced, in accordance with the present invention, by conventional site-directed or cassette mutagenesis, two avenues for routinely identifying residues of a spider silk protein which can be modified without adversely affecting particular mechanoelastic properties. See Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons 1987, 1990) (hereafter "Ausujbel”) at ⁇ 8.
  • Oligonucleotide- directed mutagenesis comprising [i] synthesis of an oligonucleotide with a sequence that contains the desired nucleotide substitution (mutation), [ii] hybridizing the oligonucleotide to a template comprising a structural sequence coding for a spider silk protein or variant and [iii] using T4 DNA polymerase to extend the oligonucleotide as a primer, is preferred because it is readily applied in determining the effect(s) of particular changes to a spider silk protein structural sequence, its relative expense may militate in favor of an alternative, known direct-mutagenesis method.
  • a spider silk variant that correspond to a portion of a spider silk protein would be a polypeptide containing the amino-acid sequence that corresponds to at least one repeating structural unit of an amorphous or crystalline domain of a spider silk protein, absent the other structural portions.
  • the ratio of amorphous and crystalline domains or subunits could be increased or decreased to provide a recombinant spider silk variant that had varied mechanostuctural properties, as desired for a particular textile, composite or ballistic material. For example, decreasing the ratio of amorphous subunits to crystalline subunits can increase the tensile strength of a spider silk variant according to the present invention.
  • crystalline and amorphous domains are those of a spider dragline silk from Nephila clavipes, wherein a crystalline domain or subunit can comprise the amino-acid sequence Ala-Gly-Ala- Gly-Ala-Gly-Ala-Gly-Tyr-Gly-Ala-Gly-Ala-Gly-Ala- Gly-Ala-Gly-Ala-Gly-Tyr-Gly-Ala-Ala-Ser-Gly-Ala- Gly-Ala-Gly-Ala-Gly-Glu-Gly-Ala-Gly-Glu-Gly-Gly- Ala-Gly-Glu-Gly-Glu-Gly-Ala-Gly-Glu-Gly-Ala-Gly-Glu-Gly-Ala-Gly- Tyr-Gly-Tyr.
  • An amorphous domain or subunit can similarly comprise the amino-acid sequence Ala-Gly- Ala-Gly-Tyr-Gly-Ala-Ala-Ser-Arg-Ile-Thr-Cys-Gly- Thr-Pro-Gly-Ser-Gly-Gln-Gly-Phe-Asp-Tyr-Arg-Ile- Arg-Arg-Glu-Gly-Tyr-Gly-Gly-Leu-Gly-Arg-Arg-Glu- Gly-Tyr-Gly-Gly-Leu.
  • An alternative amorphous domain or subunit can also comprise the amino-acid sequence Arg-Arg-Glu-Gly-Tyr-Gly-Gly-Leu-Gly.
  • a recombinant spider silk protein or variant can have an N-terminal sequence of Ile-Ser-His-Val-Pro-Thr- His-Glu-Asp-Glu-Ser-Ala-Ala-Val-Gly-Ala-Gly-Ala- Gly-Ala-Gly-Ala-Ala-Ala-Gly-Ser-Gly-Ala-Gly-Ala.
  • spider silk variants within the present invention can be fragments of the cloned and expressed molecule that retain at least one commercially useful mechanoelastic property of a spider silk protein, and that are homologous to a spider silk protein. Such fragments could be produced by known de novo-synthesis techniques and by fragmentation of the spider silk protein molecule itself, as well as by producing a genetically- engineered vector/host cell system that expresses a spider silk protein fragment encoded by a heterologous polynucleotide used to transform the host.
  • a polynucleotide molecule encoding a spider silk protein or a spider silk protein variant would preferably comprise a nucleotide sequence, corresponding to a desired amino-acid sequence, that is optimized for the host cell of choice (see below) in terms of codon usage, initiation of translation and expression of recoverable amounts of a commercially useful spider silk protein or a spider silk protein variant.
  • the vector selected for transforming a chosen host organism with such a polynucleotide molecule should allow for efficient maintenance and transcription of the sequence encoding the polypeptide.
  • Vectors can be used for cloning and expression of a polynucleotide according to the present invention in a host cell.
  • Such vectors can be derived, for example, from a bacteria, a virus, a filamentous phage (such as a M-13 derived phage) , a cosmid, a yeast or a plant.
  • Vectors will preferably include a replicator, a selectable marker, and a cloning site. See Ausubel at ⁇ 1.51. Protocols for obtaining and using such vectors are known to those in the art. Ausujel at ⁇ 1.5-1.15, 9.1-9.6 and 13.4-13.11.
  • bacteria-derived vectors examples include plas id vectors such as pBR322, pUC19, pSP64, pUR278 and pORFl. Ausubel at ⁇ 1.5.
  • suitable viral vectors are those derived from phage, vaccinia, retrovirus, baculovirus, or a bovine papilloma virus.
  • phage vectors include ⁇ + , AEMBL3, A2001, AgtlO, ⁇ gtll, Charon 4a, Charon 40, and ⁇ ZAP/R. See id. at ⁇ 1.10-1.12.
  • pKB3 and pSCll are exemplary of vaccinia vectors (see, e.g., Chakrabarti et al., Molec. Cell. Biol. 5:3401-9 (1985) and Mackett et al J. Virol. 49:857-
  • Suitable retroviral vectors can also be used. See Hollis et al., Nature 296:321-325 (1982) .
  • baculovirus vectors are pAcRP23, described in Matsuura, J. Gen. Virol. 68:1233-50
  • filamentous phage vector is an M13-derived vector like M13mpl8,
  • yeast vector examples include Ylp5,
  • Trichoderma reesei Trichoderma reesei . Ausubel , ⁇ 13.4-13.6.
  • bovine papilloma-derived vectors can be used. See, e.g., Dimaio et al Proc. Nat'l Acad.
  • vectors are preferably selected that express such long polynucleotides efficiently.
  • Suitable vectors for expressing such a polynucleotide include those that efficiently express a polynucleotide that is greater than 11 kb, e.g., phage vectors such as EMBL3 or gtll or yeast vectors such as 2/xm plasmid or pYACC3. See Ausubel at ⁇ 1.10 and 13.4.
  • DNA that encodes a spider silk protein or variant can be isolated and expressed in a recombinant microbe or other organism, by known procedures, to produce the desired polypeptide in commercially useful amounts.
  • Such a DNA can be isolated by screening nucleic acid libraries generated from microorganisms expressing a spider silk protein or variant according to the present invention. See Ausubel at ⁇ 5 and 6.
  • oligonucleotide probes that are complementary to a polynucleotide encoding, e.g., a portion of the N-terminus or known domains of a spider silk protein or variant of the present invention, e.g., as a crystalline domain comprising a portion of the amino acid sequence depicted in Figure 4, such as GGC-GAA-GGC-GCT-GGC- GAG-GGC-GGT-GCT; an amorphous domain comprising a portion of an amino acid sequence depicted in Figure 1, such as GGA-TAT-GGC-GGT-CTA-GGA or an alternative amorphous domain comprising an amino-acid sequence Arg-Arg-Glu-Gly-Tyr-Gly-Gly-Leu-Gly.
  • the spider silk protein has an N-terminal sequence of Ile-Ser-His-Val-Pro-Thr-His- Glu-Asp-Glu-Ser-Ala-Ala-Val-Gly-Ala-Gly-Ala-Gly- Ala-Gly-Ala-Ala-Ala-Gly-Ser-Gly-Ala-Gly-Ala. See Ausubel at ⁇ 6.
  • a probe as a template for generating other probes useful for isolating a spider silk protein or variant-encoding polynucleotide according to the present invention, e.g., based on the N-terminal, crystalline or amorphous domain sequences described above.
  • Such a probe can be used by known procedures for screening a genomic or cDNA library as described above, or as a basis for synthesizing PCR probes for amplifying a cDNA generated from an isolated RNA encoding a spider silk protein or variant according to the present invention.
  • transformants can be selected for expression by a host cell of a spider silk protein or variant, by use of selection media appropriate to the vector used, RNA analysis or by the use of antibodies specific for a spider silk protein according to the present invention. See, e.g., Ausubel at ⁇ 9.5.2 (selectable markers), ⁇ 9.8 (RNA analysis), ⁇ 10.6-8 (detection of proteins) , ⁇ 11.1- 1.2 (immunoassays) and ⁇ 11.3-.16 (preparation and use of monoclonal, polyclonal and antipeptide antibodies for protein detection) .
  • a cDNA can then be cloned into a suitable expression vector and used to transform a host cell, as described below. See Ausubel at ⁇ 15.4.
  • Suitable host cells in this context include prokaryotic cells (bacterial or blue-green algal) and eukaryotic cells such as yeast, insect, mammalian and human cells.
  • Suitable host cells of the present invention can preferably include microorganisms, e.g., of the genera Aeromonas, A ⁇ pergillu ⁇ , Bacillis, Escherichia, Kluyveromyce ⁇ , Pichia, Rhodococcu ⁇ , Saccharomyce ⁇ and Streptomyce ⁇ .
  • microorganisms used as host cells include, as bacterial cells, E. coli and Bacillu ⁇ , and as yeast cells, Saccharomyce ⁇ cerevisiae strains X2181-1B, with genotype ⁇ trpl gall adel his2 (available from the Yeast Genetic Stock Center, Berkeley, California); strain ATCC 52683, with genotype a his2 adel trpl metl4 ura 3 (aka strain "J17,” available from the American Type Culture Collection, Rockville, Maryland) ; and strain ATCC 46183, with genotype a hisl trpl (aka “strain IL166-5B," also available from the American Type Culture Collection) .
  • yeast cells Saccharomyce ⁇ cerevisiae strains X2181-1B, with genotype ⁇ trpl gall adel his2 (available from the Yeast Genetic Stock Center, Berkeley, California); strain ATCC 52683, with genotype a his2 adel trpl metl4 ura 3 (aka
  • a polynucleotide of the present invention into a bacterial cell
  • known procedures can be used according to the present invention such as by transfection, e.g., using calcium phosphate precipitation, electroporation, DEAE dextran, pelletizing with a DNA gun or using a recombinant phage virus. See Ausubel, at ⁇ 1.8.
  • Other known procedures can also be employed to obtain a recombinant host cell that expresses a heterologous spider silk protein according to the present invention, as will be apparent to those skilled in the art.
  • the lithium acetate method exploits the fact that alkali cations make yeast cell membrane permeable to DNA; in addition, uptake of foreign DNA is promoted by the presence in the medium of a high-molecular-weight molecule, polyethylene glycol.
  • An alternative method, spheroplast transformation can be used but is more time-consuming than the lithium acetate procedure, though it results in a higher efficiency of transformation per input DNA.
  • Another type of suitable expression system of the present invention entails the use of a mammalian host cell transformed with a polynucleotide within the present invention.
  • Suitable vectors can be used that express a spider silk protein or variant efficiently in mammalian host cells such that the protein is expressed in commercially useful quantities.
  • suitable mammalian- cellular hosts which can be used for this purpose are Chinese ovary (CHO) cells as described by Urlaub & Chasin, Proc. Nat'l Acad. Sci. USA 77:4216 (1980) and baby hamster kidney (BHK) cells, exemplified by a cell line deposited under accession number ATCC CCL 10 and another line ATCC CCL 70.
  • CHO Chinese ovary
  • BHK baby hamster kidney
  • Standard methodology in this regard is detailed in Ausubel , supra , at ⁇ 9 & 13.
  • baculoviral expression systems conventional transformation procedures with pACRP- derived vectors are used to transform suitable host cells including those of, e.g., Spodoptera (such as sf9 cells), Trichoplusia , and Heliothi ⁇ . See Luckow & Summers, Biotechnology 6:47-55 (1988); Miller, Ann. Rev. Microbiol. 42:177-199 (1988); Maeda, Ann. Rev, of Microbiol. 34:351-72 (1989).
  • Spodoptera such as sf9 cells
  • Trichoplusia Trichoplusia
  • Heliothi ⁇ e.g., vaccinia viral expression systems.
  • Host cells comprising a polynucleotide which encodes a spider silk protein or variant of the present invention can be grown under conditions that provide expression of a desired polypeptide in recoverable or commercially useful amounts. See id. , ⁇ 1 and 13.
  • a silk spider protein suitable for solublization is the silk of the golden orb-weaving spider, N. clavipes , a large spider found in the tropical and subtropical areas of the western hemisphere. Moore, Am. Mid. Natur. 98:95-108 (1977). This species produces five to seven different silk proteins, but it is the major ampullate gland silk (dragline) that possesses the highest strength. Three morphological regions distinguish the major ampullate gland: the tail, the sac and the duct. The tail is the site of about 90% of the major ampullate gland's protein synthesis activity; the ampulla is a storage site for soluble dragline silk; and the duct appears to be involved with secretion and ordering of silk. Bell & Peakall, J. Cell Biol. 42:285-95 (1969).
  • Major ampullate glands of N. clav pe ⁇ ,. as in other spiders, can be dissected and messenger RNA (mRNA) from the gland isolated and purified, in accordance with the present invention, by oligo d(T) cellulose chromatography. Dragline silk cDNA can then be constructed by reverse-transcribing the gland mRNA.
  • mRNA messenger RNA
  • a portion of the amino-acid sequence of the natural protein can be determined, e.g., by Edman degradation, and synthetic oligonucleotide probes can be constructed based on this sequence information, taking into account the redundancy of the codons encoding such a protein.
  • mRNA can be isolated by known procedures (See, e.g., Jd. , at ⁇ 4) from the major ampullate gland which produce a silk protein.
  • the mRNA is then reversed transcribed to construct a cDNA library, followed by screening with the above-mentioned probes (see, e.g., Ausubel , at ⁇ 5 and 6, respectively).
  • the synthesized cDNA can be cloned into an expression vector and the lambda gtll Sfi-Not vector (available from Promega Biotech) can be employed. Lambda gtll Sfi-Not DNA is thus used as a vector for orientation-specific cDNA cloning, allowing the expression of cloned inserts as polypeptides fused with 9-galactosidase.
  • Directional cloning can be achieved by using a unique oligodeoxynucleotide primer-adapter containing the recognition site for Notl upstream from an oligo(dT) sequence to prime first strand synthesis.
  • the double stranded molecules can be digested with NotI (and EcoRI for linkers) .
  • the spider silk cDNA is ready to ligate into the provided EcoRI-NotI vector arms.
  • Recombinant phage can then be recognized by their ability to form colorless plaques when plated on lac-hosts (E. coli Y1089(r-) and Y1090(r-)) in the presence of X-GAL (5-bromo-4-chloro-3-indolyl- 3-galactopyranoside) .
  • lac-hosts E. coli Y1089(r-) and Y1090(r-)
  • X-GAL 5-bromo-4-chloro-3-indolyl- 3-galactopyranoside
  • Dragline silk clones can be further isolated by using nucleic acid probes constructed specifically from dragline silk protein sequences. Once identified, these clones can be repurified to ensure purification of only the dragline silk cDNA. Positive clones can be sequenced and the overlapping sequences can be used to determine the full length nucleic acid sequence that encodes a dragline silk protein of Nephila . See Au ⁇ ubel at ⁇ 6 and 7. The full length cDNA can then be reconstructed from the cloned fragments. Id. at ⁇ 3.16.
  • This cDNA can be expressed in E. coli or other suitable host organism, and the presence of the resultant silk/3-GAL fusion protein can be ascertained, for example, by immunoscreening, thereby to identify recombinants which produce the protein. Selected clones can then be cultured and tested, for example, by western blotting for the presence of the protein.
  • the protein can be purified in a conventional manner, either from the host medium or from a preparation of lyzed host cells, for example, by using an immunoaffinity adsorbent column.
  • Dragline spider silk protein from the species Nephila clavipe ⁇ was found to have a molecular weight of about 350,000 daltons and is encoded by a polynucleotide of about 12,000 daltons.
  • the present invention is further described by reference to the following, illustrative examples. In these examples, specimens of the following arachnid species were used: N. clavipe ⁇ Nephilinae, supplied by Angela Choate (USDA, Gainesville, Florida) ; Argiope aurantia Lucas and Neo ⁇ cona domiciliorum Hentz, supplied by Mark Stowe, (University of Florida, Gainesville) .
  • Live specimens were housed in individual cages and fed a diet of gerraan cockroaches, Blatella germanica (Blattellidae) . Some specimens were frozen in liquid nitrogen and stored at -70'C for subsequent nucleic acid extractions.
  • Silk samples (approximately 1.0-2.0mg) were 5 placed in 13x100mm sterile glass borosilicate test tubes. The solvents listed in Table 1 were added to a final concentration of l.Oug/ul and solubility determined visually at room temperature.
  • Major ampullate gland silk samples (2.0mg) were first dissolved in 2.0ml of a hydrochloric/propionic acid mixture at room temperature for 20 min with slight vortexing. Solubilized samples (100 ⁇ l at l.o ⁇ g/ ⁇ l) were vacuum dried in pyrolyzed vials and purged with argon gas. Hydrolysis was carried out by placing 200 ⁇ l of constant boiling 6N HCl in the bottom of an acid-resistant reaction vessel along with 2 sodium sulfite crystals. The vessel was again purged with argon gas, sealed under vacuum and placed at 150°C for 1 hour. Argon was used as a purging gas because of its purity and because it contributes fewer artifact peaks in the subsequent analysis. Sodium sulfite was discovered to be useful as an oxygen scavenger and aids in the recovery of cysteine, serine, and threonine (Ted Tanhauser, Cornell University, personal communication) .
  • the amino acid composition of the secretion of (MaAS) from N. clavipe ⁇ is shown in Tables 2 and 3.
  • Glycine, alanine, glutamic acid/glutamine and arginine were the most abundant amino acids, comprising 74% of all amino acids present.
  • the major ampullate gland silk has been considered for use in the production of dragline and frame threads of the web.
  • the dragline has a high tensile strength (198 grams per denier, gpd) and it has a rupture elongation of 18%, determined according to Zemlin, Technical Report 69-29-CM. AD 684333 10760-5020 (1967).
  • the composition of the material from the large ampullate gland generally agrees with the published analyses of dragline from N. clavipe ⁇ , see Zemlin, loc. cit. , and Work & Young, J. Arachnol. 15:65-80 (1987), but some differences were observed.
  • Table 3 shows the amounts of various amino acid side chains in dragline silk of N. clavipe ⁇ .
  • Dragline silk is composed predominantly of the small side-chain amino acids glycine, alanine and serine, which would allow them to conform to the antiparallel beta-pleated sheet model proposed by Pauling and Corey, Proc. Nat. Acad. Sci. 39:253-256
  • Dragline silk spider spider 2 spider 3 Trials —3 small side chains polar side chains acidic/amide side chains basic side chains cyclic imino side chain aromatic side chain sulfur containing aliphatic side chain hydroxyl side chain small side chains: gly+ala+ser polar residues: asp+thr+ser+glx+tyr+lys+his+arg acidic/amide residues: asx+glx basic side chains: lys+his+arg cyclic imino side chain: pro aromatic side chain: phe+tyr sulfur containing: met+cys aliphatic side chain: ala+val+leu+ile hydroxyl side chain: ser+thr
  • a dragline silk of Nephila was sequenced by Edman degradation, and synthetic oligonucleotide probes were constructed that corresponded to this sequence, as shown in Figure 1, taking into account the redundancy of the codons encoding such a protein.
  • a crystalline domain of the spider silk protein was thus determined to have an amino-acid sequence of Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Tyr-Gly-Ala-Gly- Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Tyr-Gly-Ala-Ala-Syr-Gly-Ala-Ala-Syr-Gly-Ala-Ala- Ser-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Glu-Gly-Ala-Gly- Glu-Gly-Ala-Gly-Glu- Gly-Ala-Gly-Glu- Gly-Ala-Gly-Glu- Gly-Ala-Gly-Glu- Gly-Ala-Gly-Tyr-Gly-Tyr.
  • the probes were constructed by known methods. See Au ⁇ ubel at ⁇ 6.3- 6.4.
  • Glandular dissection was employed to obtain mRNA encoding a dragline spider silk protein.
  • Major ampullate glands were dissected out of living spiders through a 1.5 cm longitudinal incision along the ventral abdomen. The glands were removed carefully to avoid degradation of the luminar contents. The glands were immediately transferred to a medium containing 0.10M sodium chloride and 0.015M sodium citrate (SSC) .
  • SSC sodium citrate
  • Protease inhibitors phenylmethyl sulfonyl fluoride (PMSF) at a final concentration of 6-10mg/ml (Methods in Enzv ology 26:3-27 (1972)), and 20 units/ml of aprotonin (Piperno et al., Proc. Natl. Acad.
  • the synthesized cDNA was cloned into an expression vector, the lambda gtll Sfi-Not vector (commercially available, e.g., from Promega Biotech, Promega Corporation, Madison, WI) was employed.
  • Lambda gtll Sfi-Not DNA is a vector designed for orientation specific cDNA cloning which allows the expression of cloned inserts as polypeptides fused with beta-galactosidase.
  • Directional cloning was achieved by using a unique oligodeoxynucleotide primer-adapter containing the recognition site for NotI upstream from an oligo(dT) sequence to prime first strand synthesis.
  • Dragline silk clones were isolated by using nucleic acid probes constructed specifically from dragline silk protein sequences. Once identified, these clones were repurified 3x to ensure purification of only the dragline silk cDNA. Positive clones were sequenced by conventional procedures and the overlapping sequences used to determine the full length nucleic acid sequence that encodes a dragline silk protein of Nephila . See, e.g., Ausubel at ⁇ 6 and 7. Next, the full length cDNA was reconstructed from the cloned fragments and was determined to have a length of 12 kb. Jd. at ⁇ 3.16.
  • the cloned silk was expressed in E. coli via the vector described in Example 5 above, and the resultant silk/0-Gal fusion protein was screened with a Protoblot immunoscreening system by screening phage plaques containing the recombinant protein and by western blotting of lambda lysogens after purification on a protosorb lacZ immunoaffinity adsorbent column.
  • the purification on the LacZ immunoaffintiy column was alternatively used to elute the silk protein, cleaved from the lacZ protein by treatment with cyanogen bromide, using increased salt concentration. The cyanogen bromide and salt were then removed from the purified silk protein by dialysis.
  • a variant of a spider silk protein can be generated by modification of a cDNA encoding a spider silk protein as described in Example 5, above, by known site directed or cassette mutagenesis techniques according to, e.g., Ausubel at ⁇ 8. Suitable fragments of the cDNA can be modified by site directed or cassette mutagenesis to provide cDNA that encodes a dragline silk protein variant having a at least one of each of the amorphous and crystalline domains depicted in
  • a spider silk protein variant can be expressed that has a molecular weight of about 110,000, wherein the variant comprises about 10 crystalline and 10 amorphous domains, as depicted in Figures 4 and 2, respectively.
  • EXAMPLE 8 CLONING AND EXPRESSION OF A SPIDER SILK MUTEIN
  • a mutein of a spider silk protein can be generated by modification of a cDNA encoding a spider silk protein as described in Example 5, above, by known site directed or cassette mutagenesis techniques as described by, e.g.,
  • Suitable portions of a cDNA can be modified by site directed or cassette mutagenesis to provide cDNA that encodes a dragline silk protein mutein having a at least one of each of the amorphous and crystalline domains depicted in Figures 2 and 4, respectively.
  • a spider silk protein mutein can be expressed that has a molecular weight of about 85,000 wherein the mutein comprises about 10 crystalline and 5 amorphous domains, as depicted in Figures 4 and 2, respectively.
  • Such a mutein would have higher tensile strength than an endogenous spider silk protein because the ratio of crystalline domains to amorphous domains would be greater than 1.
  • a cDNA is expressed according to Example 6, above, and a spider silk protein mutein can be expressed that has a molecular weight of about 80,000 wherein the mutein comprises about 5 crystalline and 10 amorphous domains, as depicted in Figures 4 and 2, respectively.
  • a mutein would have higher elasticity than an endogenous spider silk protein because the ratio of crystalline domains to amorphous domains would be less than 1.

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Abstract

On peut obtenir des protéines de soie arachnéennes recombinées sous forme commerciale par clonage et par l'expression dans une cellule hôte d'un polynucléotide codant une protéine de soie arachnéenne endogène ou une variante de celle-ci. Un procédé de solubilisation d'une protéine de soie arachnéenne permet le séqençage d'une protéine de soie arachnéenne.
PCT/US1991/002222 1990-04-19 1991-03-29 Proteines de soie arachneennes recombinees obtenues par genie genetique Ceased WO1991016351A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0452925A3 (en) * 1990-04-20 1992-07-29 The University Of Wyoming Isolated dna coding for spider silk protein, a replicable vector and a transformed cell containing the isolated dna, and products thereof
WO1994029450A3 (fr) * 1993-06-15 1995-02-09 Du Pont Nouveaux analogues de soie d'araignee produits par recombinaison
WO1997008315A1 (fr) * 1995-08-22 1997-03-06 Basel Richard M Procedes de clonage servant a obtenir des proteines de soie d'araignee extremement resistantes
US5728810A (en) * 1990-04-20 1998-03-17 University Of Wyoming Spider silk protein
FR2774588A1 (fr) * 1998-02-11 1999-08-13 Oreal Composition cosmetique ou dermatologique contenant au moins une proteine de soie d'arachnides naturelle, recombinante ou un analogue
WO2001090389A3 (fr) * 2000-05-25 2002-06-06 Du Pont Production de proteines du type soie chez les plantes
WO2011039345A1 (fr) 2009-10-02 2011-04-07 Vib Vzw Protéines de soie de tétranyque
EP3434768A1 (fr) 2007-03-16 2019-01-30 Empresa Brasileira De Pesquisa Agropecuária Embrapa Protéines dérivées des toiles d'araignées néphilengys cruentata, avicularia juruensis et parawixia bistriata
CN110551194A (zh) * 2019-09-19 2019-12-10 天津大学 重组蜘蛛卵鞘丝蛋白复合物及其生成的人造卵鞘丝
WO2020112742A1 (fr) * 2018-11-28 2020-06-04 Bolt Threads, Inc. Purification alcaline de protéines de soie d'araignée
WO2020145363A1 (fr) * 2019-01-09 2020-07-16 Ｓｐｉｂｅｒ株式会社 Fibroïne modifiée
EP4194488A1 (fr) * 2021-12-09 2023-06-14 Samsung Electronics Co., Ltd. Polypeptide, composition de photorésine l'incluant et procédé de formation de motif l'utilisant
CN117304018A (zh) * 2023-09-25 2023-12-29 烟台泰和新材高分子新材料研究院有限公司 一种利用丙酰氯副产物连续高效生产丙酸钙的方法

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USRE21454E (en) * 1936-01-20 1940-05-21 Fibroin spinning solutions
GB2162190A (en) * 1984-07-06 1986-01-29 Pa Consulting Services Improvements in or relating to production of silk

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USRE21454E (en) * 1936-01-20 1940-05-21 Fibroin spinning solutions
GB2162190A (en) * 1984-07-06 1986-01-29 Pa Consulting Services Improvements in or relating to production of silk

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Title
CHEMICAL AND ENGINEERING NEWS, issued 25 July 1988, "Spider Silk Gene Route to High Terrible Fiber", pages 24 and 25. *
PROCEEDING OF THE NATIONAL ACADEMY OF SCIENCES, Vol. 87, issued September 1990, XU et al., "Structures of a Protein Superfiber: Spider Dragline Silk", pages 7120-7124. *
TECHNICAL REPORT, 69-29-CM, AD 684333, U.S. Army Natick, 01760-5020, issued 1968, ZEMLIN, "A Study of the Mechanical Behaviour of Spider Silk", pages 1-65. *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5728810A (en) * 1990-04-20 1998-03-17 University Of Wyoming Spider silk protein
EP0452925A3 (en) * 1990-04-20 1992-07-29 The University Of Wyoming Isolated dna coding for spider silk protein, a replicable vector and a transformed cell containing the isolated dna, and products thereof
US5989894A (en) * 1990-04-20 1999-11-23 University Of Wyoming Isolated DNA coding for spider silk protein, a replicable vector and a transformed cell containing the DNA
EP0452925B1 (fr) * 1990-04-20 2000-02-09 The University Of Wyoming ADN codante pour la protéine de la soie d'araignée, vecteur et cellule transformée la contenant, et produits
US6268169B1 (en) 1993-06-15 2001-07-31 E. I. Du Pont De Nemours And Company Recombinantly produced spider silk
WO1994029450A3 (fr) * 1993-06-15 1995-02-09 Du Pont Nouveaux analogues de soie d'araignee produits par recombinaison
EP1413585A3 (fr) * 1993-06-15 2004-08-04 E.I. Du Pont De Nemours And Company Nouveaux analogues de soie d'araignée produits par recombination
WO1997008315A1 (fr) * 1995-08-22 1997-03-06 Basel Richard M Procedes de clonage servant a obtenir des proteines de soie d'araignee extremement resistantes
US6841162B2 (en) 1998-02-11 2005-01-11 L'oreal Cosmetic or dermatological composition contacting at least one natural or recombinant spider silk or an analog
EP0943323A1 (fr) * 1998-02-11 1999-09-22 L'oreal Composition cosmétique ou dermatologique contenant au moins une protéine de soie d'arachnides naturelle, recombinante ou un analogue
FR2774588A1 (fr) * 1998-02-11 1999-08-13 Oreal Composition cosmetique ou dermatologique contenant au moins une proteine de soie d'arachnides naturelle, recombinante ou un analogue
US7148039B2 (en) 1998-02-11 2006-12-12 L'oreal Cosmetic or dermatological composition contacting at least one natural or recombinant spider silk or an analog
WO2001090389A3 (fr) * 2000-05-25 2002-06-06 Du Pont Production de proteines du type soie chez les plantes
US6608242B1 (en) 2000-05-25 2003-08-19 E. I. Du Pont De Nemours And Company Production of silk-like proteins in plants
US6965060B2 (en) 2000-05-25 2005-11-15 E. I. Du Pont De Nemours And Company Production of silk-like proteins in plants
EP3434768A1 (fr) 2007-03-16 2019-01-30 Empresa Brasileira De Pesquisa Agropecuária Embrapa Protéines dérivées des toiles d'araignées néphilengys cruentata, avicularia juruensis et parawixia bistriata
US8501913B2 (en) 2009-10-02 2013-08-06 Vib Vzw Spider mite silk proteins
WO2011039345A1 (fr) 2009-10-02 2011-04-07 Vib Vzw Protéines de soie de tétranyque
WO2020112742A1 (fr) * 2018-11-28 2020-06-04 Bolt Threads, Inc. Purification alcaline de protéines de soie d'araignée
WO2020145363A1 (fr) * 2019-01-09 2020-07-16 Ｓｐｉｂｅｒ株式会社 Fibroïne modifiée
US12319718B2 (en) 2019-01-09 2025-06-03 Spiber Inc. Modified fibroin
CN110551194A (zh) * 2019-09-19 2019-12-10 天津大学 重组蜘蛛卵鞘丝蛋白复合物及其生成的人造卵鞘丝
CN110551194B (zh) * 2019-09-19 2023-03-21 天津大学 重组蜘蛛卵鞘丝蛋白复合物及其生成的人造卵鞘丝
EP4194488A1 (fr) * 2021-12-09 2023-06-14 Samsung Electronics Co., Ltd. Polypeptide, composition de photorésine l'incluant et procédé de formation de motif l'utilisant
US12441769B2 (en) 2021-12-09 2025-10-14 Samsung Electronics Co., Ltd. Polypeptide, photoresist composition including the same, and method of forming pattern using the same
CN117304018A (zh) * 2023-09-25 2023-12-29 烟台泰和新材高分子新材料研究院有限公司 一种利用丙酰氯副产物连续高效生产丙酸钙的方法

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