WO2004016651A2

WO2004016651A2 - Spider silk polypeptide

Info

Publication number: WO2004016651A2
Application number: PCT/GB2003/003578
Authority: WO
Inventors: Simon Mcqueen Mason; Natalia Pouchkina
Original assignee: University of York
Current assignee: University of York
Priority date: 2002-08-15
Filing date: 2003-08-15
Publication date: 2004-02-26
Anticipated expiration: 2005-02-15
Also published as: AU2003260719A1; WO2004016651A3; AU2003260719A8; GB0218977D0

Abstract

This invention describes the isolation and sequencing of a new silk polypeptide and includes vectors, transgenic plants and animals expressing DNA molecules encoding said polypeptide and methods for the production of said silk polypeptides.

Description

Spider Silk Polypeptide

This invention relates to isolated DNA molecules encoding spider silk polypeptides; spider silk polypeptides; vectors and transgenic plants and animals expressing said DNA molecules; and methods for the production of said silk polypeptides.

Silk production by arthropods has evolved over millions of years. Arthropod silk proteins have advantageous physical properties which are envied by industry involved in the synthesis of high performance polymers. Silk use is most highly developed in Arachnids, particularly spiders.

An orb web, the typical spider web, is constructed of as many as six types of silk, each composed primarily of protein and exhibiting different mechanical properties. Each type of silk is secreted and stored by specialized epithelial cells in different types of abdominal gland until it is extruded by tiny spigots on the spinnerets. The silk proteins are used singly, or in combinations for draglines, retreats, egg sacs or prey- catching snares.

The major ampullate silk or "dragline silk" is an extracellular semicrystalline polymer, which, besides forming the dragline, is used to form the frame of the web. Whilst each gossamer strand is one tenth the width of a human hair, the silk must perform functions such as absorbing the energy of a flying insect at speeds of up to 20 miles per hour so that the prey neither breaks nor bounces off the trap, and support the weight of the spider.

The most studied dragline silk is from Nephila clavipes, the golden orb weaving spider. The combined high tensile strength (4 x 109 N/m2) and elasticity (35%) translates into a toughness that is superior to all manmade or natural fibres, including silkworm threads. The silk is five times stronger than steel, yet 30% more flexible than nylon and can absorb three times the impact force without breaking than the super-filaments Kevlar™ (Dupont) and Spectra™ (AlliedSignal)(Gosline et al, Endeavour 10, 37-43, 1986). In addition to the tensile strength and elasticity of the silk, the additional characteristics of supercontraction, insolubility and non-allergenicity make the use of silk in medical and industrial applications an attractive option. Medical applications include; artificial tendons/ligaments, wound-closure systems, such as vascular wound repair devices, hemostatic dressings, patches, glues and sutures. Industrial applications include; bullet-proof vests, light-weight body armour, cables, ropes and parachute cords, even fishing line.

Other advantages in using spider silk rather than the synthetic alternatives such as Kevlar™ is that this fibre is derived from petrochemicals, which are processed under extreme, often polluting conditions and add to the waste accumulating in landfills.

The reproduction of synthetic fibres that mimic spider silk has proved technologically difficult. Whilst synthetic fibres such as Kevlar™ do contain the crystalline regions embedded in a rubbery, amorphous matrix similar to the structure of the spider silk, obtaining this structure, and the outstanding fibre properties it imparts, requires spinning long polymer chains at high temperatures and pressures using strongly acidic organic solvents. This further adds to the waste problem.

The spider has overcome the problem of spinning these long polymer chains by using a starting material referred to as 'dope'. This is a liquid-crystalline solution containing the silk protein molecules, which are initially coiled but which during the later stages of spinning become uncoiled and aligned with each other and along the axis of the developing fibre, whilst the solution moves through the insects continuously narrowing spinning organ.

Despite its superior mechanical properties, spider silk is not used commercially because of a constraint on supply. Efforts to farm arachnids have failed due to their territorial nature.

Progress has been made in the cloning and expression of spider silk proteins leading to the development of technologies to mass produce recombinant dragline silks {Prince et al, Biochemistry 34:10979-100885,1995). Xu et α/.,(Proc. Natl. Acad. Sci 87, 7120, 1990) reported the sequence for a portion of the repetitive sequence of dragline protein, Spidroin 1 f om Nephila clavipes based on a partial cDNA clone. This protein consists of a repetitive unit, which is a maximum of 34 amino acids long and is not rigidly conserved. The repeat unit is composed of three different segments; (i) a 6 amino acid segment that is conserved in sequence but has deletions of 3 or 6 amino acids in many of the repeats; (ii) a 13 amino acid segement dominated by a polyalanine sequence of 5-7 amino acids (iii) a 15 amino acid highly conserved segment, predominantly a Gly-Gly-Xaa repeat with Xaa being alanine, tyrosine, leucine or glutamine. A second identified dragline-silk protein, Spidroin 2, exhibits an entirely different repetitive motif than Spidroin 1, consisting of linked beta-turns in proline-rich regions which alternate with beta-sheet regions composed of polyalanine segments (Hinman & Lewis, J.B.C. 267 (27):19320;1992). The dragline silk proteins range in size from 272kDa-750kDa

US5728810 discloses the isolated cDNA coding for Spidroinl and 2 from Nephila clavipes and further discloses a replicable vector containing cDNA which encodes for spider silk protein and which is capable of expressing spider silk protein and a transformed cell or microorganism containing cDNA which codes for spider silk protein which is capable of expressing spider silk protein.

Although successful expression of spider silk has been demonstrated in prokaryotes such as E.coli, significant problems remain, such as instability of the sequences used due to recombination and rearrangement in the repetitive areas of the gene, inefficient transcription, translational pausing and premature termination resulting in limitations on the length of the silk that can be produced efficiently (less than or equal to 1000 amino acids), low protein yields (4-300 mg/liter) and low solubility of the produced fibre. Improvements in the production of the silk proteins were made when the genes were expressed in the methyltropic yeast Pichia pastoris, with increases in the length of the protein (>1600 amino acids), increased yields (663mg/liter), but only 15% was soluble in the yeast lysate.

WO99/47661 (Nexia Biotechnology) discloses a method for the recombinant production of biofilaments, such as spider silk, using transgenic animals, rodents, ruminants or goats. In this method a nucleic acid sequence encoding a biofilament from Nephila and Aranus species under the control of a promoter directs the expression of a polypeptide in milk-producing or urine-producing cells. By exploiting anatomical similarities between spider silk glands and mammalian glands, it has proved possible to develop genetically modified goats whose milk contains dissolved spider silk proteins. Several problems still however exist. The extraction and concentration of the proteins from the milk is proving to be technologically difficult. The milk requires repeated treatment with solvents in order to precipitate the proteins, which and further processed by filtration, dialysis and/or chromatography to obtain a pure product. It also remains unknown whether the proteins exist in the natural state, and furthermore, the expression of dragline silk in its natural state may not be particularly useful as exposure to water turns it into a rubbery mess.

The use of a plant for the production of silk or silk-like proteins has a number of advantages over a microbial expression system. For example, as a renewable source, a plant expression system requires far less energy and material consumption than microbial methods. Similarly, a plant expression systems represent a far greater available biomass for protein production than a microbial system. Zhang et al (Plant Cell Rep. 1996, 16 3-4, 174-79) teach the expression of an elastin-based protein polymer in transgenic tobacco plants, whilst WO 01/90389 and WO 01/94393 teach methods for the production of silk and silk-like proteins in green plants.

It is therefore desirable to identify new silk proteins and expression systems which do not suffer from the problems associated with prior art silk proteins and their heterologous production.

According to an aspect of the invention there is provided an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence consisting of the sequence as represented in

Figure la or 2a; ii) a nucleic acid sequence which encodes a polypeptide domain comprising the amino acid sequence AGRGQGGYGQGAGG and at least two motifs rich in polyalanine wherein said polyalanine motifs comprise at least 6 alanine amino acid residues; iii) a nucleic acid sequence which hybridises to the sequence presented in

(i) and (ii) above and which encodes a silk polypeptide; and iv) a nucleic acid sequences that are degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) - (iii) above.

In a further preferred embodiment of the invention said domain further comprises a motif comprising a repetitive tripeptide sequence, GGX (glycine:glycine: X is any amino acid residue), and at least one SS(serine: serine)-polyalanine sequence.

In a preferred embodiment of the invention said nucleic acid molecule anneals under stringent hybridisation conditions.

Stringent hybridisation/washing conditions are well known in the art. For example, nucleic acid hybrids that are stable after washing in 0.1xSSC,0.1% SDS at 60°C. It is well known in the art that optimal hybridisation conditions can be calculated if the sequence of the nucleic acid is known. Typically, hybridisation conditions uses 4 - 6 x SSPE (20x SSPE contains 175.3g NaCl, 88.2g NaH₂PO₄ H₂O and 7.4g EDTA dissolved to 1 litre and the pH adjusted to 7.4); 5-1 Ox Denhardts solution (50x Denhardts solution contains 5g Ficoll (Type 400, Pharmacia), 5g polyvinylpyrrolidone and 5g bovine serum albumen); lOOμg-l.Omg/ml sonicated salmon herring DNA; 0.1-1.0% sodium dodecyl sulphate; optionally 40-60% deionised formamide. Hybridisation temperature will vary depending on the GC content of the nucleic acid target sequence but will typically be between 42°- 65° C.

■ In a further preferred embodiment of the invention said nucleic acid molecule is a cDNA.

In an alternative preferred embodiment said nucleic acid molecule is genomic DNA.

In a preferred embodiment of the invention said nucleic acid molecule is isolated from an arachnid, preferably a spider. Preferably said spider is selected from the genus Euprosthenops. ssp., preferably said spider is Euprosthenops spidroin. According to a further aspect of the invention there is provided a polypeptide encoded by a nucleic acid molecule according to the invention.

Preferably said polypeptide comprises the amino acid sequence motif AGRGQGGYGQGAGG and is a silk protein.

In a preferred embodiment of the invention said polypeptide is a variant polypeptide wherein said variant polypeptide varies from the sequence presented in Figure lb or 2b which sequence has been modified by deletion, addition or substitution of at least one amino acid residue. Preferably said modification modifies the physical properties of said polypeptide.

A variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations which may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants which retain or enhance the same biological function and activity as the reference polypeptide from which it varies.

A functionally equivalent polypeptide(s) according to the invention is a variant wherein one in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues, or one in which one or more amino acid residues includes a substituent group. Conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between amide residues Asn and Gin; exchange of the basic residues Lys and Arg; and replacements among aromatic residues Phe and Tyr. In addition, the invention features polypeptide sequences having at least 75% identity with the polypeptide sequences as hereindisclosed, or fragments and functionally equivalent polypeptides thereof. In one embodiment, the polypeptides have at least 85% identity, more preferably at least 90%> identity, even more preferably at least 95%o identity, still more preferably at least 97% identity, and most preferably at least 99%o identity with the amino acid sequences illustrated herein.

It will be apparent that modifications which enhance the mechanical properties or which facilitate the heterologous expression of said polypeptide are particularly desirable. For example, modifications which address different codon usage between species; modifications which enhance the stability or solublity of said polypeptides.

In a preferred embodiment of the invention said polypeptide comprises the amino acid sequence represented in Figure lb or 2b. Preferably said polypeptide consists of the amino acid sequence presented in Figure lb or 2b.

According to a further aspect of the invention there is provided a vector including a nucleic acid molecule according to the invention. Preferably said vector is adapted for recombinant expression.

A vector including nucleic acid (s) according to the invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome for stable transfection.

Preferably the nucleic acid in the vector is operably linked to an appropriate promoter or other regulatory elements for transcription in a host cell. The vector may be a bi- functional expression vector which functions in multiple hosts.

By "promoter" is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.

Constitutive promoters include, for example CaMV 35S promoter (Odell et al (1985) Nature 313, 9810-812); rice actin (McElroy et al (1990) Plant Cell 2: 163-171); ubiquitin (Christian et al . (1989) Plant Mol. Biol. 18 (675-689); pEMU (Last et al (1991) Theor Appl. Genet. 81: 581-588); MAS (Velten et al (1984) EMBO J. 3. 2723- 2730); ALS promoter (U.S. Application Seriel No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid- responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellie et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilised. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al (1996) Plant Physiol. 112(3): 1331- 1341; Van Camp et al (1996) Plant Physiol. 112(2): 525-535; Canevascni et al (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al (1993) Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al (1993) Proc. Natl. Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.

Particularly preferred promoters are seed specific promoters of which USP (Baumlein H, et al. (1991) Mol. Gen. Genet. 225: 459- 467) is an example.

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.

In a preferred embodiment the promoter is an inducible promoter or a developmentally regulated promoter.

According to a further aspect of the invention there is provided a cell transfected or transformed with a nucleic acid molecule or vector according to the invention.

Preferably said cell is a eukaryotic cell.

In a preferred embodiment of the invention said cell is selected from the group consisting of; a fungal cell (e.g Pichia spp, Saccharomyces spp, Neurospora spp); insect cell ( e.g Spodoptera spp); mammalian cells (e.g. COS cell, CHO cell); a plant cell.

In a preferred embodiment of the invention said cell is a plant cell.

According to a further aspect of the invention there is provided transgenic plant comprising a cell according to the invention.

In a preferred embodiment of the invention said plant is selected from the group consisting of; corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuas), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Iopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus' spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia inter grifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.

In a further preferred embodiment of the invention said plant is Nicotiana spp.

According to a further aspect of the invention there is provided a cell culture comprising a cell according to the invention.

According to a further aspect of the invention there is provided a seed comprising a cell according to the invention.

According to a further aspect of the invention there is provided a transgenic non- human animal comprising a cell according to the invention.

In a preferred embodiment of the invention said transgenic non-human animal is selected from the group consisting of; goat, cow, sheep.

According to a further aspect of the invention there is provided a silk fibre comprising a polypeptide according to the invention.

According to a further aspect of the invention there is provided material comprising a silk fibre according to the invention.

According to a further aspect of the invention there is provided a cell culture vessel comprising a cell according to the invention. Preferably said vessel is a bioreactor. According to a yet further aspect of the invention there is provided a method for production of a silk protein; i) providing a cell culture according to the invention, ii) providing conditions conducive to the growth of said cell culture; and optionally iii) extracting silk protein, or variants thereof, from said cell culture or cell culture media.

In a preferred method of the invention said cell culture is comprised in a bioreactor.

According to a further aspect of the invention there is provided a method for the production of silk protein from a transgenic plant.

In a preferred method of the invention said method comprises the steps of: i) providing a plant cell according to the invention; ii) regenerating said cell into a plant; and optionally iii) extracting silk protein, or variants thereof from said plant.

According to a further aspect of the invention there is provided a method for the production of silk protein from a non-human transgenic animal.

In a preferred method of the invention said method comprises the steps of:

i) providing a transgenic non-human animal according to the invention; and ii) extracting silk protein, or variants thereof from said animal.

In a preferred embodiment of the invention said method extracts silk protein from said animals milk.

An embodiment of the invention will now be provided by example only and with reference to the following figures Figure la represents the DNA sequence of cDNA clone MaSPl clone 14; Figure lb is the deduced amino acid sequence of clone 14;

Figure 2a represents the DNA sequence of cDNA clone MaSPl clone 3; Figure lb is the deduced amino acid sequence of clone 3;

Materials and Methods

Spiders - Adult Euprosthenops spiders were captured in Kenya and maintained under laboratory conditions in clear Perspex boxes. Every third day all spiders were fed flies (Musca domestica) and their webs were sprayed with tap water.

Genomic DNA isolation and cloning of the non-repetitive 3 '-end of Euprosthenops spidroin 1 gene — Genomic DNA was isolated from cephalothorax (head and legs) of a single female spider using GenElute ™ Mammalian Genomic DNA Miniprep Kit (Sigma) following manufacture's instructions. In order to obtain 3 '-end sequence of Euprosthenops spidroin 1 gene, shown to be conserved among a number of species (Beckwitt and Arcidiacono, 1994), a polymerase chain reaction (PCR) was performed with primers based on known 3 '-end sequence for Nephila clavipes spidroin 1 gene;

SIR - 5'-GGCGAATTCACCTAGGGCTTGATAAACTGATTGAC-3' (Xu and Lewis, 1990).

S1L - 5'-CCCGGATCCGGAGGTGCCGGACAAGGAGGATATGGAGGT-3' (Xu and Lewis, 1990).

Genomic DNA served as a template and Clontech's Advantage™ Polymerase Mix was used. PCR conditions were as follows: (94°C - lmin) x 1; (94°C - 30 sec, 68°C - lmin, 72°C - 2 min) x 30; (72°C - 5 min) x 1. 10 μl of PCR reaction mix was run on 1%) agarose gel, a single band of about 400 bp was isolated from the gel using Qiaex II Agarose Gel Extraction kit (Qiagen), subcloned into Invitrogen pCR2.1-TOPO vector and sequenced at MWG sequencing facility, Germany.

Euprosthenops cDNA synthesis - Major ampullate glands were dissected from the abdomen of a single female Euprosthenops spider in an ice-cold Cupiennius Ringer solution (13.03 g NaCl, 0.51 g KC1, 0.89 g CaCl2, 1.04 g MgCl₂ x 6H₂O - pH 8.2). 5μ/ml RNasin Inhibtor (Promega) was added to the solution prior to use. Total RNA was isolated from the glands using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) following manufacture's instructions. Due to the use of a total RNA as a starting material, first-strand cDNA synthesis reaction was performed according to Clontech SMART™ Library Construction protocol

(http://www.clontech.com/smart/index.shtml). A specific 3'- primer was used instead of an oligo-dT primer (CDS III/3 '-primer in Clontech SMART™ Library Construction Kit) in the first-strand synthesis reaction driven by Invitrogen ThermoScript RNase H^" reverse transcriptase. The primer composition was based on known 3 '-end sequence for Nephila clavipes spidroin 1 gene:

SIR - 5'-GGCGAATTCACCTAGGGCTTGATAAACTGATTGAC-3' (Xu and Lewis, 1990).

This was followed by conventional second-strand synthesis procedure where the reaction components required (5x second- strand buffer and 20x second-strand enzyme cocktail) were taken from PCR-Select™ cDNA subtraction kit (http://www.clontech.com/pcr-select/index.shtml).

Cloning of a partial cDNA sequence encoding the repetitive region of Euprosthenops major ampullate gland spidroin 1 — 1:5 diluted second-strand cDNA synthesis mix served as a template and Invitrogen ThermalAce Polymerase was used in subsequent PCR reactions. Primer compositions were as follows. SIR (Xu and Lewis, 1990) was used as a 3' primer. 5' primer (5 ALA) was based on a polyalanine stretch sequence identified at the very beginning of the non-repetitive 3 '-end region of Euprosthenops spidroin 1 gene.

5 ALA - 5' -GCTGCCGCAGCAGCAGCT - 3' (current study).

PCR conditions were as follows: (98°C - 3min) x 1; (98°C - 30 sec, 61°C - 30 sec, 72°C - 3 min) x 30; (72°C - 10 min) x 1. 10 μl of PCR reaction mix was separated onl% agarose gel, various size PCR products ranging from 500 to 1000 bp were isolated from the gel using Qiaex II Agarose Gel Extraction kit (Qiagen), subcloned into Invitrogen pCR2.1-TOPO vector and sequenced at MWG sequencing facility, Germany.

Northern analysis - For comparative Northern analysis total RNA was isolated from major ampullate glands and cephalothorax of adult female Euprosthenops and Nephila senegalensis spiders using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) following manufacture's instructions. About 10 μg of total RNA was used for each loading. RNA was separated on formaldehyde gel, transferred to a Hybond-N membrane according to standard procedure (rtttp://www.MolecularCloning.com and consequently hybridised with the following P-labelled RNA templates. Linearised pCR2.1-TOPO vector (Invitrogen) containing a 400 bp non-repetitive 3 '-end region of the Euprosthenops spidroin 1 gene served as a template for the first riboprobe synthesis. The second probe was prepared from a linearised pCR II-TOPO vector (Invitrogen) containing a 600 bp partial cDNA sequence encoding a putative repetitive region of Euprosthenops spidroin 1 gene. Both probes were generated using Promega Riboprobe^R in-vitro transcription system following the manufacturer's instructions. RNA loading control experiments were performed with a riboprobe made with partial cDNA sequence for putative Nephila senegalensis V-type ATPase (Pouchkina et al., submitted).

EXAMPLE 1

To expand the database of known spidroin sequences we obtained a number of partial cDNA clones encoding repetitive region of Euprosthenops major ampullate gland spidroin las well as a genomic clone encoding its C-terminal.

Like previously published fibroin sequences from spiders (Guerette et al., 1996; Hayashi and Lewis, 1998), our sequences encode a repetitive alanine- and glycine- rich protein. Assembled repeats from a number of cDNA clones were aligned and a consensus ensemble repeat was generated, results not shown.

Euprosthenops MaSpl-like AGRGQGGYGQGAGGNAΛAΛAAAAAAAAΛAGQGGQGGQGQGGYGQGAGSSAAAAAAAΛΛ Nephila MaSpl

AGRGGLGGQGAGAAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGSQG

Euprosthenops MaSpl silk sequences show the same type of conserved repeat motifs to those seen in orb weaving spiders in that the repeats are made up of regions containing AGRGQGGYGQGAGG followed by a poly alanine stretch and this followed in turn by a GGX region. There are however, also striking differences between the repeat structure or Euprosthenops and that of Nephila MaSpl. Most notably, the Euprosthenops silk protein repeat contains an additional polyalanine stretch, not seen in orb weaver silk proteins. This second polyalanine strech is preceded by two serine residues another feature not seen in the Nephila protein, h addition to having an extra polyalanine strech, the polyalanine stretches themselves are notably longer than those seen in Nephila MaSPl, containing 7-14 alanines in series compatred to 4 to 7 in Nephila sp. (Xu and Lewis, 1990). This is consistent with the data showing Euprosthenops silk to be stronger and stiffer than silks of other spiders (Madsen et al, 1999; Vollrath and Knight, 2001) and contained a greater amount of β -conformations and higher degree of crystallinity than did the dragline silk of nephila sp. (Shao et al., 1999). In the same context, it is also notable that the GGX region of the ensemble repeat is considerably shorter (18 residues) in Euprosthenops, compared to 28 in Nephila MaSPl. These hypothetical random coil regions are believed to underlie the elasticity of the silk, which is reported to be much lower in Euprosthenops (Madsen et al., 1999).

From the data gathered the ensemble Euprosthenops repeats are not rigidly conserved. Alanine-rich regions could be interrupted by a single substitution from alanine to threonine, an amino acid which is rare in araneoid fibroins (Gatesy et al., 2001). The transition of alanine (GCX) to threonine (ACX) is generated by a single nucleotide substitution (from G to A). Single or coupled alanine to valine, serine or glycine changes are also observed within the polyalanine regions. Present study undoubtedly expands our understanding of relationship between the sequence of the spider silk and its mechanical properties. The retention of a number of amino acid motifs among araneoid and non-araneoid fibroins implies that the sequences themselves are critical to understanding of the elasticity and strenght of different silk fibres. Together with the evident impact of the silk fibre spinning mechanism (Clavert, 1998; Vollrath, 1999) amino acid composition of the silk fibres is likely to have the dominant influence on fibre formation and properties.

References

Beckwitt, R. and Arcidiacono, S. 1994. Sequence conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae). JBiol Chem. 269, 6661-3.

Clavert, P. 1998. Nature 393, 309.

Coddington, J. and Levi, H. 1991. Ai inu. Rev. Ecol. Syst. 22, 565.

Engster, M.S. 1976. Studies on silk secretioni n the Trichoptera (F.Limnephilidae). Cell Tissue Research 169, 77-92.

Gatesy, J., Hayashi ,C, Motriuk, D., Woods, J. and Lewis, R. 2001. Extreme diversity, conservation, and convergence of spider silk fibroin sequences. Science 291, 2603-5.

Gosline, J.M., Guerette, P.A., Ortlepp, C.S., Savage, K.N. 1999. The mechanical design of spider silks: from fibroin sequence to mechanical function. J. Exp. Biol. 202 , 3295-303.

Guerette, P.A., Ginzinger, D.G., Weber, B.H. and Gosline , J.M. 1996. Silk properties determined by gland-specific expression of a spider fibroin gene family. Science 111, 112-5. Hayashi, C.Y.and Lewis, RN. 1998. Evidence from flagelliform silk cDNA for the structural basis of elasticity and modular nature of spider silks. J. Mol. Biol. 275, 773- 784.

Jelinski, L.W., Blye, A., Liivak, O., Michal, C, LaVerde, G., Seidel, A., Shah, N. and Yang, Z. 1999. Orientation, structure, wet-spinning, and molecular basis for supercontraction of spider dragline silk. Int. J. Biol. Macromol. 24, 197-201.

Kaplan, D.L., Lombardi, S.J., Muller, W.S., Fossey, S.A. 1991. Silk. In Biomaterials: Novel materials from biological sources: Byrom D., Ed.; Stockton press: New York.

Madsen , B., Shao, Z.Z., Vollrath, F. 1999. Variability in the mechanical properties of spider silks on three levels: interspecific, intraspecific and intraindividual. Int. J. Biol. Macromol. 24, 301-6.

Pouchkina, N. N., Stanchev, B.S. and McQueen-Mason, S.J. From EST Sequence to Spider Silk Spinning: Identification and Molecular Characterisation of Nephila senegalensis Major Ampullate Gland Peroxidase NsPox (submitted).

Shao, Z., Young, R.J., Vollrath ,F. 1999. The effect of solvents on spider silk studied by mechanical testing and single-fibre Raman spectroscopy. Int. J. Biol. Macromol. 24, 295-300.

Vollrath, F., Knight ,D.P. 2001. Liquid crystalline spinning of spider silk. Nature 410, 541-8.

Xu , M., Lewis, RN. 1990 Structure of a protein superfiber: spider dragline silk. Proc. Natl .Acad .Sci. USA 87, 7120-4.

Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule consisting of the nucleic acid sequence as represented in Figure la or 2a; ii) a nucleic acid molecule which encodes a polypeptide domain comprising the amino acid sequence AGRGQGGYGQGAGG and at least two motifs rich in polyalanine wherein said polyalanine motifs comprise at least 6 alanine amino acid residues; iii) a nucleic acid molecule wherein said domain in (ii) further comprises a motif comprising a repetitive tripeptide sequence, GGX (glycine:glycine: X is any amino acid residue), and at least one SS(serine:serine)-polyalanine amino acid sequence.

2. An isolated nucleic acid molecule according to Claim 1 wherein said nucleic acid molecule is a cDNA.

3. An isolated nucleic acid molecule according to Claim lor 2 wherein said molecule is isolated from an arachnid.

4. An isolated nucleic acid molecule according to Claim 3 wherein said arachnid is a spider of the genus Euprosthenops.

5. An isolated nucleic acid according to Claim 4 wherein said spider is

Euprosthenops spidrion.

6. A polypeptide encoded by a nucleic acid molecule according to any of Claims 1- 5.

7. A polypeptide which comprises a polypeptide domain comprising the amino acid sequence AGRGQGGYGQGAGG and at least two motifs rich in polyalanine wherein said polyalanine motifs comprise at least 6 alanine amino acid residues and said polypeptide is a silk protein.

8. A polypeptide according to Claim 7 wherein said polypeptide is a variant polypeptide which variant polypeptide varies from the sequence presented in Figure lb or 2b which sequence has been modified by deletion, addition or substitution of at least one amino acid residue.

9. A polypeptide according to Claim 8 wherein said polypeptide comprises the amino acid sequence represented in Figure lb or 2b.

10. A polypeptide according to Claim 9 wherein said polypeptide consists of the amino acid sequence presented in Figure lb or 2b.

11. A vector including a nucleic acid molecule according to Claims 1-5.

12. A vector according to Claim 11 wherein said vector is adapted for recombinant expression.

13. A vector according to Claim 12 wherein said vector is provided with a promoter which is operably linked to said nucleic acid molecule.

14. A cell transfected or transformed with a nucleic acid molecule according to any of Claims 1-5 or vector according to any of Claims 11-13.

15. A cell according to Claim 14 wherein said cell is a eukaryotic cell.

16. A cell according to Claim 15 wherein said cell is selected from the group consisting of; a fungal cell; an insect cell; a mammalian cell; a plant cell.

17. A cell according to Claim 16 wherein said cell is a plant cell.

18. A plant comprising a cell according to Claim 17.

19. A cell according to Claim 14 wherein said cell is a prokaryotic cell.

20. A cell culture comprising a cell according to any of Claims 14-17 or 19.

21. A seed comprising a cell according to Claim 17.

22. A transgenic non-human animal comprising a mammalian cell according to Claim 16.

23. A transgenic non-human animal according to Claim 22 which is selected from the group consisting of: a goat; a cow; or a sheep.

24. A silk fibre comprising a polypeptide according to any of Claims 6-10.

25. Material comprising a silk fibre according to Claim 24.

26. Use of a silk polypeptide according to any of Claims 8-12 for the manufacture of material.

27. A cell culture vessel comprising a cell according to any of Claims 14-17 or 19.

30 A vessel according to Claim 27 wherein said vessel is a bioreactor.

31. A method for production of a silk protein; i) providing a cell culture vessel according to Claim 27. ii) providing conditions conducive to the growth of a cell culture contained in said vessel; and optionally iii) extracting silk protein, or variant proteins thereof, from said cell culture or cell culture media.

32. A method according to Claim 31 wherein said cell culture is comprised in a bioreactor.

33. A method for the production of silk protein from a transgenic plant comprising the steps of: i) providing a plant cell according to Claim 17; ii) regenerating said cell into a plant; and optionally iii) extracting silk protein, or variants thereof from said plant.

34. A method for the production of silk polypeptide from a non-human transgenic animal comprising the steps of: i) providing a transgenic non-human animal according to Claim 22 or 23; and ii) extracting silk protein, or variants thereof, from said animal.

35. A method according to Claim 34 wherein said silk polypeptide is extracted from said animals milk.