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WO1997008315A1 - Procedes de clonage servant a obtenir des proteines de soie d'araignee extremement resistantes - Google Patents

Procedes de clonage servant a obtenir des proteines de soie d'araignee extremement resistantes Download PDF

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Publication number
WO1997008315A1
WO1997008315A1 PCT/US1996/013767 US9613767W WO9708315A1 WO 1997008315 A1 WO1997008315 A1 WO 1997008315A1 US 9613767 W US9613767 W US 9613767W WO 9708315 A1 WO9708315 A1 WO 9708315A1
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Prior art keywords
dna
silk
protein
spider
primers
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PCT/US1996/013767
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English (en)
Inventor
Richard M. Basel
Glenn R. Elion
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Basel Richard M
Elion Glenn R
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Publication date
Application filed by Basel Richard M, Elion Glenn R filed Critical Basel Richard M
Priority to AU71529/96A priority Critical patent/AU7152996A/en
Priority to JP9510520A priority patent/JPH11511325A/ja
Priority to IL12339896A priority patent/IL123398A0/xx
Priority to EP96932937A priority patent/EP0848754A1/fr
Priority to BR9612625A priority patent/BR9612625A/pt
Publication of WO1997008315A1 publication Critical patent/WO1997008315A1/fr

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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

  • This invention relates to novel methods of producing DNA fragments encoding for spider silk proteins.
  • the present invention also relates to the DNA sequences encoding the spider silk proteins.
  • This invention still further relates to novel methods of producing spider silk proteins using the above-described DNA sequences.
  • the invention also relates to methods of purifying these spider silk proteins and manufacturing fibers and films from them.
  • Clones developed by the methods of the present invention produce commercially useful quantities of high molecular weight spider silk proteins ranging in molecular weights from 90,000 to over 250,000, which are from 40% to greater than 100% of the molecular weight of natural major ampulate (dragline) spider silk protein obtained from Nephila clavipes. Because the silk made from these high molecular weight proteins have superior physical properties, such as high tensile strength and substantial elasticity, the cloned silk proteins of the present invention are of considerable industrial importance. These spider silk proteins have been cloned by several methods of the present invention and the natural sequence spider silk clones have been produced in E. coli expression systems.
  • spider silk proteins which have been expressed at levels in excess of 2 grams per liter of cell mass. These spider silk proteins are then purified and used for many purposes such a ⁇ spinning fibers, forming films and other applications resulting from the weaving of filaments.
  • Silk production by many diverse animal orders e.g.. insects, arachnids and mites
  • Spiders for example, produce natural webs and draglines having high tensile strengths.
  • Silkworms on the other hand, although producing silks at high production rates, have silk proteins that are considered inferior to spider silk proteins in their physical properties. For example, silkworm proteins have considerably lower tensile strengths than spider silk proteins.
  • Orb weavers and other spiders although naturally producing low quantities of silk filaments (less than economic for commercialization), have strong filaments. In fact, spider filaments can be several times stronger than Kevlar * (9.5 x IO 4 vs 3 x IO 4 Jkg "1 ) .
  • spider silk protein filaments are a preferred choice for parachutes, sails, body armor and other high strength applications requiring strong filaments. Additionally, these spider filaments find utility as absorbent films for many heavy metals and organics including biological weapons. They also find utility as absorbents that selectively bind DNA and absorbents for many other chemicals, flavors and fragrances. Although it might be hypothesized that spider silk could be produced from culturing spiders, this is impractical for several reasons. First, in addition to being very difficult to raise, spiders will eat their neighbors if grown in very high densities. Second, spiders produce only small amounts of silk protein making production of even milligram quantities prohibitively expensive. As a result of these limitations, the only acceptable method for producing commercial quantities of spider silk proteins is to clone the spider gene into an acceptable large scale production vector. The present invention accomplishes that objective.
  • Synthetic silk protein genes have previously been produced by making short base pair segments and then using large numbers of repeating unit ⁇ . Proteins with modest molecular weights (ranging from 20,000 to 80,000) have been obtained by such a process. To achieve a variety of physical properties, this process has been varied and synthetic proteins with different sequences have been produced. For example, prior workers have used sequences obtained by taking small lengths of naturally occurring silk proteins and changing the sequences.
  • one of tne objects of the present invention is to overcome the above-mentioned problems that occur with polymerized short DNA sequences. This is accomplished with the present invention by the production of long DNA that encode for high strength, high molecular weight silk proteins. Because of the potential that high strength ma ⁇ or ampulate (dragline) spider silk offers, silks from orb weavers such as Nephila clavipes have been studied in attempts to understand the molecular basis of their strength. researchers have also attempted with limited success to clone the natural protein or make a synthetic silk gene by incorporating the repetitive elements responsible for the high strength of spider silk fibers.
  • Spider silk proteins typically are 200,000 kDa or higher and the corresponding genes also have at least one intron. As such, it i ⁇ projected that the size of the DNA fragment would be in the range of 5-10 Kb plus any introns. With current technology, genes of this magnitude are still notoriously difficult to clone.
  • the present invention has overcome this problem.
  • Nephila clavipes dragline silk was taken by Xu et al. (Proc. Natl. Acad. Sci. 87:7120, 1990) .
  • Xu et al. ascertained a portion of the repetitive sequence of a spider dragline silk from a partial clone. Although this repeating unit encoded for up to 34 amino acids, it was not exactly conserved as the sequence had deletions and changes in some of the repeats. Nevertheless, Xu et al. discovered two important areas in the sequence -- repetitive regions which give spider silk some of their properties and a non-repetitive (carboxy) region. Hinman and Lewis (J: Biol. Chem.
  • the present invention relates to the novel synthesis of partial and full length spider silk protein clones. Some of these partial length clones have also been multimerized into other clones with molecular weights up to and exceeding those of natural spider silk.
  • the present invention has made it possible to develop natural silk-like clones that have a complete range of properties.
  • One skilled in molecular biology can use these clones as a starting point for creating clones with other useful silk properties such as strength, yield point, adhesiveness and plasticity.
  • these new sequences can be used as starting points to design other synthetic genes. For some spiders which incorporate colors or pigments into their silk proteins, these methods may also permit naturally colored protein.
  • the present invention also relates to unique chemical methods for fermentation of transfected hosts in culture media.
  • One of the major problems of producing silk proteins by bacterial fermentations is the partial ⁇ iytotlon of proteins by proteases.
  • the rate of protein decomposition from proteases can in some cases overcome the rate of high molecular weight silk protein expression, thereby making commercial operations impractical.
  • the present invention overcomes this potential problem.
  • Figure 1 shows the 2Kb DNA sequence for encoding the spider silk protein.
  • This invention relates to a proces ⁇ of producing DNA fragment ⁇ encoding for silk protein, comprising the step ⁇ of (i) selecting target DNA harvested from a silk-producing spider, the target DNA comprising a plurality of repetitive and non-repetitive regions; (ii) selecting a single strand DNA primer of at least 10 nucleotides having a DNA sequence that is complementary to a region in the target DNA; and (iii) repetitively combining the DNA primer with melted target DNA and incubating the combined DNA primer and target DNA with nucleotides and a DNA polymerase having proofreading ability to produce the DNA fragment, wherein the DNA fragment i ⁇ complementary to said target DNA and is at least 2 Kb.
  • DNA fragments of at least 5 Kb can be produced.
  • the process comprises the step of using two different DNA primers instead ⁇ l out.
  • the target DNA is cDNA made by reverse transcription of full length mRNA coding for spider silk, and the proces ⁇ further comprises the steps of (i) adding a primer site to the amino terminal end of the first strand cDNA made thereof and (ii) using the poly T region of the cDNA a ⁇ a fir ⁇ t polymera ⁇ e priming region.
  • a second primer site is created at the unknown end of the DNA using a ligation cassette.
  • a second primer site is created at the unknown end of the DNA using a terminal transferase to make a primer site selected from the group con ⁇ isting of poly dT, poly dA, poly dG and poly dC.
  • the DNA primer for the above-described processe ⁇ of producing a DNA fragment can be ⁇ elected from DNA repre ⁇ ented by ⁇ tarting and ending sequences (i) - (xx) given below:
  • N G, A, T, C
  • V G, A, C
  • B G, T, C
  • H A, T, C
  • D G, A, T
  • K G, T
  • S G, C
  • W A, T
  • M A, C
  • Y C, T
  • R A, G.
  • the target DNA is selected by hybridization to a DNA probe, having at least one of the above-described sequences (i) - (xx) , that is reversibly bound to a support to enrich for the silk- encoding DNA fragments.
  • the process comprises the steps of (i) selecting a target DNA encoding silk protein harvested from a silk-producing spider, the target DNA comprising a plurality of repetitive and non-repetitive regions; (ii) selecting a first pair of different DNA primers, the first pair of DNA primers both being complementary to a region in the target DNA, and at least one of the first pair of DNA primers being represented by the sequences (i) - (xxvi) ; (iii) producing a first DNA fragment by repetitively combining the first pair of DNA primers with melted target DNA and incubating the combined DNA primers and target DNA with nucleotides and a DNA polymerase having proofreading ability to produce the first DNA fragment, the first DNA fragment being complementary to the target DNA and at least 2 Kb.
  • This multimerizat. on process further comprises the steps of (iv) selecting a second pair of different DNA primers, at least one of the second pair of DNA primers being different than both of the sequences of the first pair of DNA primers, and at least one of the second pair of DNA primers being represented by the sequences (i) - (xxvi) ; (v) producing a second DNA fragment by repetitively combining the second pair of DNA primers with melted target DNA and incubating the combined DNA primers and target DNA with nucleotides and a DNA polymerase having proofreading ability to produce the second DNA fragment, the second DNA fragment being different than the first DNA fragment and also being complementary to the target DNA, the second DNA fragment being at least 2 Kb; (vi) restricting the first and ⁇ econd DNA fragment ⁇ ; and (vii) recombining the restricted portions of the first and second DNA fragments into a multimerized DNA, the multimerized DNA encoding spider silk protein and being at lea ⁇ t 4 Kb in length.
  • all DNA primers are represented by ⁇ equence ⁇ (i) - (xxvi) .
  • all DNA primer ⁇ are different.
  • the multimerized DNA is at least 6 Kb or 8 Kb in length.
  • thi ⁇ invention relate ⁇ to a DNA sequence encoding spider silk protein, wherein the DNA sequence comprise ⁇ a plurality of repetitive and non-repetitive region ⁇ and has a length of at least 2 Kb. In a more particular embodiment, the DNA sequence has a length of at least 5 Kb. In a still more particular DNA sequence embodiment of the present invention, the DNA comprises the sequence illustrated in Figure 1.
  • this invention comprises the step ⁇ of (i) ⁇ electing a DNA; (ii) inserting the DNA into an expres ⁇ ion vector; (iii) transfecting host cells with the expression vector; (iv) fermenting the transfected host in culture media to produce silk protein; and (v) recovering the silk protein.
  • the culture media for fermenting the transfected host contains protease inhibitor.
  • the process comprise ⁇ the steps of (i) applying ultrasound energy to rupture the host cells; (ii) applying ultrasound energy to resuspend the silk protein; and (iii) centrifuging the ruptured host cell ⁇ to separate cell membranes from the silk protein.
  • purification of the silk protein is accomplished by ultrafiltration or alcohol precipitation.
  • this invention relates to a process comprising the steps of (i) concentrating silk protein purified by ultrafiltration or alcohol precipitation; (ii) drawing a fiber of concentrated silk protein; (iii) spinning silk fibers to produce a ⁇ ilk thread; and (iv) washing the silk thread to remove any solubilization reagents.
  • the solubilization reagents are selected from the group consisting of hexafluoroisopropanol, sodium hydroxide, potassium hydroxide, urea, urea phosphate, lithium salts, organic solvents, guanidine nydrochloride, ammonium sulfate, acetic acid, phosphoric acid, dichloroacetic acid, formic acid and sulfuric acid.
  • the process further comprise ⁇ the ⁇ tep of coating the silk fiber or thread with oxides of tin or titanium.
  • the present invention relates to a fabric comprising the spider silk threads made according to any of the processes of the invention.
  • the fabric comprises spider silk threads made in accordance with any of the processes of the present invention in combination with Kevlar ® , graphite or carbon fibers, as well as silkworm silk.
  • the protein can be used a ⁇ a coating, extruded into a fiber, or made into a polymeric film.
  • spiders may have up to eight kinds of silk glands. Although no spider species has all eight silk glands, all spiders have at lea ⁇ t three such glands and most have five. Each gland produces a different type of silk having different properties. For example, some silk dries quickly, while other silk remains sticky.
  • Spiders belong to the phylum Arthropoda. class Arachnida and order Araneae. True spiders belong to the suborder Labidognatha. Other spider types include comb footed, crab, fisher, funnel web, hackled-band, orb weavers, jumping and ogre faced stick. Spiders from any of the following genus groups can be used in accordance with the present invention: Micrathena. Mastophora. Metepeira, Araneus, Argiope. Nephila or Gas eraca L a.
  • Orb weaver ⁇ are among the mo ⁇ t successful spider groups because they have evolved silk ⁇ with remarkable ⁇ trength and flexibility.
  • the orb weaver ⁇ are known a ⁇ Argionidae and include: arrowheaded shaped Micrathena sagittata. bolas spider Mastophora cornigera, labyrinth Metepeira labyrinthea, marbled Araneu ⁇ marmoreu ⁇ . black-and-yellow garden Argiope bruennichi. golden silk Nephila clavipes. and spiny bodied Gasteracantha cancrif ⁇ rmis.
  • Nephila clavipe ⁇ has been studied the most in genetic research since its silk thread ⁇ are strong and its silk glands are large and easy to dissect. Other orb weavers also produce strong silk threads.
  • spiders While all spiders produce silk, the proteins that form the silk threads vary considerably in their molecular makeup and serve a variety of purposes. For example, the Antrodiatus ⁇ piders spin a simple kind of silk comprising just two proteins. In contrast, spiders in the family Araneoidea, called web spinners, produce up to eight different kinds of silk. Orb weavers produce a variety of silks using several proteins to create webs of greater ⁇ trength and flexibility.
  • Spider ⁇ ilk proteins also have different qualitie ⁇ depending upon which silk gland it was spun from.
  • the stronge ⁇ t silks known are from the major ampulate gland of orb weavers.
  • the major ampulate (dragline) silk was selected for this work because of its physical strength and non-sticky properties.
  • This dragline silk is composed of protein although carbohydrates are associated with the fiber.
  • the liquid silk undergoes an irreversible transition to an insoluble lorm composed OJ. a nigh relative ratio of alanine and glycine.
  • This fiber consists of an antiparallel /3-sheet with elastic interspaces.
  • the amino acid compo ⁇ ition of this silk (shown in the table below) mimics the composition of clones of the present invention.
  • the present inventors ⁇ et out to overcome the ⁇ e shortcomings and found that by using somewhat degenerate primer ⁇ either one or a number of PCR products could be produced.
  • Genomic DNA taken from Nephila clavipe ⁇ abdomen ⁇ was used. To isolate the DNA from the spider, the preparation method described in Sambrook et al. , Molecular Cloning: A Laboratory Manual, Vol.1-3, Cold Spring Harbor Laboratory, New York (1989) , was followed exactly. This procedure resulted in high molecular weight genomic DNA in excess of 2 Kb.
  • the inventors experimented with many primers that were related to the ⁇ equence data disclosed by Xu et al.
  • primer sequences (i) - (xx) Some of the primers used are disclosed above as primer sequences (i) - (xx) . Although these primers were also tried by Beckwith & Arcidiacono, the present inventors are the first to produce spider silk protein up to 2 Kb in length using a two primer PCR cloning system. The present inventors were also able to produce spider silk proteins with higher Kbs by the claimed cDNA and ⁇ ingle site cloning methods de ⁇ cribed below.
  • Nephila clavipes DNA isolated by the procedure of Beckwith & Arcidiacono was used along with the following two primers: primers (i) GGCGAATTCGGATCCATGGCAGCAGCAGCAGCAGCAGCT, and (ii) GGCGAATTCACCCTAGGGCTTGATAAACTGATTGAC.
  • Primer (i) codes for a poly-alanine repeat sequence based on che forward reading frame. Leader sequences that insert an in-frame start codon and both BamH I and EcoR I leader restriction sites for cloning as overhangs were also put into the primer.
  • Primer (ii) is a PCR primer (bp 2218 to 2242) based upon the reverse sequence of Xu et al. This sequence also has an in frame stop codon and an EcoR I restriction site. As shown in this Example and Example 2, the re ⁇ ults depend on the PCR conditions and are not positive without newer polymerases. The regular Taq and the Taq extender did not give the same results, presumably due to misreading or false priming.
  • the PCR mix was as follows: 5 ⁇ l Taq extender buffer (Stratagene) ; 1 ⁇ l of Taq polymerase 5 ⁇ g/ ⁇ l (Stratagene) ; 1 ⁇ l of l ⁇ g/ ⁇ l DNA template (spider genomic DNA) ; 1 ⁇ l of 2 ⁇ M primer (i) in water; 1 ⁇ l of 2 ⁇ M primer (ii) in water; 5 ⁇ l of NTP's (2 ⁇ M each of dATP, dCTP, dGTP, and dTTP, pH 7.0); 45 ⁇ l of Taq extender (Stratagene) ; and water to a total of 100 ⁇ l total.
  • the PCR cycler conditions were as follows: initial dwell 94°C. for 2 min; and PCR conditions (30 cycles) : annealing at 60°C. for 1 min.; extension at 72°C. for 2 min.; and denaturation at 94°C. for 1 min.
  • the PCR conditions of annealing at 60°C. for l min. and extension at 72°C. for 2 min. can be replaced with a treatment of 72°C. for 2 min.
  • the genomic DNA was isolated from freeze dried spider abdomens which were ground in a mortar and pestal and extracted according to Sambrook et al. , Molecular Cloning: A Laboratory Manual Vol. 1-3, Cold Spring Harbor Laboratory, New York (1989) .
  • primer (iii) GCATGCACGCATGGTGCATGGATGC
  • primer (ii) GGCGAATTCACCCTAGGGCTTGATAAACTGATTGAC primer (iii) was made from the peptide sequence 4 described by Mello et al. , Silk Polymers, ACS, Symposium, Ser 544 (1994) .
  • Primer (ii) was made as described in Example 1 above.
  • PCR mix 5 ⁇ l 10X Takara LA PCR buffer; 5 ⁇ l Takara dNTP mix; 1 ⁇ l primer (iii) (2 ⁇ M) ; 1 ⁇ l primer (ii) (2 ⁇ M) ; l ⁇ l Takara Ex Taq with proofreading activity; 1 ⁇ l spider genomic DNA; water to a total of 50 ⁇ l; and 50 ⁇ l mineral oil.
  • the Takara LA PCR buffer, dNTP mix, and Takara Ex Taq were supplied with a Takara Roll kit distributed by Panvera Corp., 565 Science Dr., Madison, WI 53711. PCR cycler conditions were as follows: initial dwell 94°C.
  • Restriction enzymes were also ⁇ imilarly u ⁇ ed to dige ⁇ t the insert.
  • the restriction protocol was as follows: 5 ⁇ g or less of plasmid or insert DNA; 5 ⁇ l of restriction enzyme 10X buffer; 5 ⁇ l 1 mg/ml acetylated BSA; 5 ⁇ l restriction enzyme (EcoR I) ; water to a final volume of 50 ⁇ l; and incubate for 3 hr. at 37°C.
  • the vector was also treated after phenol extraction and cleanup with EcoR I restriction enzyme.
  • the vector was similarly treated with calf intestinal alkaline phosphatase (CIAP) . This treatment prevented the vector from re-annealing.
  • CIP calf intestinal alkaline phosphatase
  • the CIAP protocol which was done in addition to the restriction protocol, was as follows: 10 ⁇ l CIAP 10X buffer consi ⁇ ting of 500 mM tri ⁇ -HCl, pH 9.0, 10 mM MgCl 2 , 1 mM ZnCl 2 and 10 mM spermidine; 1 unit CIAP; water to final volume of 100 ⁇ l; and incubate for 60 min. at 37°C.
  • One CIAP unit will hydrolyze 6.0 mM of p-nitrophenyl phosphate per minute at 37°C. These units are measured in a 0.1 M glycine buffer at pH 10.4 containing 1.0 mM ZnCl 2 , 1.0 M MgCl 2 .
  • the next step was to ligate the insert into the pUC18.
  • the DNA was repurified with phenol extraction and ethanol precipitation and then ligated according to the protocol described below.
  • Ligation protocol 100 ng vector DNA; 100 ng or less insert DNA; l unit T4 DNA ligase (Weiss Units) ; l ⁇ l ligase 10X buffer; water to a final volume cf 10 ⁇ l; and incubate for 1 hr. at room temperature.
  • the new vector was then inserted into EX. coli XLl MRF ' obtained from Clonetech Laboratories, Inc., 4030 Fabian Way, Palo Alto, CA 94303, using the Clonetech method for inserting supercompetent cells.
  • the transformants were selected by ampicillin resistance in LB broth 10 g/1 bactopeptone, 5 g/1 yeast extract, and 5 g/1 NaCl using 50 ⁇ g/ ⁇ l of ampicillin.
  • Clones were checked for the proper insert by first looking for the proper size of plasmid, approximately 4.3 Kb.
  • the insert was also checked by using biotinylated probes and as ⁇ aying for hybridization.
  • the be ⁇ t 5 inserts from transformation were checked for expression of the inserted protein as it was inserted in such a way that it should express within pUC18.
  • the 2 Kb insert was easily made using the PCR technique described above. This technique produced superior results over the following three methods: screening of shotgun clone libraries for silk by probes based upon peptide sequencing (Xu et al.); cDNA inserts from the silk gland (Hinman and Lewis) ; and PCR using Taq polymerase or other polymerases with no proof reading. (Beckwith and Arcidiacono) .
  • Example 2 The PCR technique of Example 2 compared to the above three methods was fa ⁇ t, did not induce error ⁇ into the sequence as was apparent from the other reported methods, and was directed only to the gene of interest. With just a little of the sequence from the amino end and carboxy end of the spider silk, this technique could be applied to the sequencing of silks other than the major ampulate (dragline) silk or to other spiders having similar properties.
  • the next step in the development process was to convert the mR ⁇ A to a good first strand template and then reliably replicate the D ⁇ A.
  • cD ⁇ A cycle kit L1310-01 obtained from Invitrogen Corp., 3985 B Sorrento Valley Blvd., San Diego, CA 92121, and a PCR amplification ⁇ ⁇ e proved unsatisfactory because the primers developed were only good for amplifying small pieces of mR ⁇ A.
  • the inventors thereafter decided to develop their own technique for obtaining a 10 Kb mR ⁇ A. The first part of this process was to optimize the reverse transcriptase reaction.
  • the preferred reverse transcriptase for making the first strand was discovered by trying variou ⁇ reverse transcripta ⁇ e enzymes, including AMV (Avian Myelobastosis Virus) reverse transcriptase (M5101) and M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase (M5301) which is modified to remove the ribonuclease H activity. See Tanese & Goff, Prec. Natl. Acad. Sci. U.S.A. 85:1977 (1988) . Both M5101 and M5301 were obtained from Promega Corp., 2800 Wood ⁇ Hollow Road, Madi ⁇ on, WI 53711.
  • mRNA has a poly A end
  • a poly T primer was used.
  • a marker ⁇ equence wa ⁇ needed and numerous pos ⁇ ibilitie ⁇ exi ⁇ ted. While putting a marker ca ⁇ ette on each end worked, that technique had a low probability of Iigating on to the low number first strand DNA. Since mRNA has a poly A end adjacent to where the carboxy end of the protein is coded, a method to label one end wa ⁇ already available. Therefore, a method that would just label the one end was adopted and a terminal transfera ⁇ e was used.
  • the preferred method is to use tne enzyme terminal transferase to add poly A at the 3 ' end of the first strand. This was done by allowing a single primer method to amplify both end ⁇ of the cDNA from the mRNA.
  • the protocol is as follows: 10 ⁇ l terminal transferase buffer (Promega formula) ; 1 ⁇ l terminal transferase (Promega) ; 5 ⁇ l of the first strand DNA from reverse transcription procedure described above; 1 ⁇ l oligo d(T) 6 ., 2 ; 1 ⁇ l d(A); and 7 ⁇ l water; and incubate for 1 hr. at 37°c. Both the terminal transferase buffer and the terminal transferase were obtained from, Promega Corp. , 2800 Woods Hollow Rd. , Madison, WI 53711-5399, catalog no. M1871.
  • the DNA was then reisolated using phenol and ethanol precipitation, and PCR was used.
  • the technique which is described below, yielded DNA strands with a poly dA strand on one end and a poly dT on the opposite end.
  • the PCR amplification of cDNA was as follows: 1 ⁇ l DNA from the terminal transferase procedure described above; 10 ⁇ l 10X Takara LA buffer; 10 ⁇ l dNTPs (Takara); 1 ⁇ l poly d(T) 20 primer; 1 ⁇ l Takara Ex Taq LA polymera ⁇ e; 78 ⁇ l water; and 100 ⁇ l mineral oil.
  • the PCR conditions were as follows: the initial dwell was 94°C. for 1 min.; the amplification cycle ⁇ (40) were: 94°C. for 30 sec; 55°C. for 2 min.; and 72°C. for 3 min.; followed by post dwell at 2°C.
  • the amplification initially showed a streak with multiple mRNA.
  • the cDNA from the initial amplification was amplified first with only primer (ii) of the 2 Kb coding for the non-repetitive region of the silk protein, which also incorporates the stop codon using 1 ⁇ l of the cDNA from the first PCR.
  • This new primer only amplifies cDNA coding for silk protein. This produce ⁇ a ⁇ elective library for silk proteins. This also gave a streak that amplified preferentially the cDNA from the silk protein.
  • Positive transformant ⁇ were assayed for insertion by checking the size of insertion with a 1% agarose gel.
  • the positive inserts were then tested for the correct insert by using PCR and poly d(T) 20 primer.
  • the positives were also tested by the antibody methods discu ⁇ ed below.
  • the positives passing the antibody tests for large mRNA were tested using SDS electrophoresis gels and found to give three different proteins also proving multiple start sites.
  • One protein was slightly larger than the 2 Kb piece and the other two proteins were slightly shorter than native ⁇ pider silk dragline protein. It was difficult, however, to get these high molecular weight proteins to stain with a Western stain, but this was also true with the native proteins.
  • primer (ii) is unique because it codes for the carboxy end of the major ampulate (dragline) silk protein. Nevertheless, it was necessary to develop a method that would get further into the amino direction and hopefully pull out the whole sequence. Two such approaches were developed. One was to use a shotgun method to make DNA clones, which is discussed below. It was believed unlikely that one would be able to clone the whole gene in one insert and make protein by this method. Because the inventors knew that the carboxy end was unique for other spider silks of interest, they believed a method could be developed for PCR which only had to start with one known unique site. This technique, which is the second approach, involved Iigating ca ⁇ settes to the end of the DNA, although the use of a terminal transferase would have been as effective.
  • the restriction digestion procedure is as follows: 2 ⁇ l 1 ⁇ g/ ⁇ l genomic DNA; 20 units of an appropriate restriction enzyme (corresponding to one of the six above-mentioned restriction cassettes or others provided the same Restriction Cassette is used with the restriction enzyme) ; 5 ⁇ l 10X buffer for restriction enzyme; distilled water up to a total of 50 ⁇ l; and incubate at 37°C. for 3 hr.
  • This restriction digest is then cleaned and reconcentrated by ethanol precipitation and redissolved in sterile water.
  • the cassette is then ligated to the respective DNA digest.
  • the ligation reaction procedure i ⁇ as follows: 5 ⁇ l genomic DNA digest; 2.5 ⁇ l of an appropriate cassette such as cassettes 1-6 mentioned above) (20 ng/ ⁇ l) ; 7.5 ⁇ l Takara ligation solution; and incubation for 3.0 min. at room temperature.
  • Thi ⁇ ligation reaction mix is then cleaned and reconcentrated by ethanol precipitation and redissolved in 5 ⁇ l of sterile water. Because the Taq in Takara's kit did not have proofreading activity or high fidelity, reagents and polymera ⁇ e from the Takara LA PCR kit were u ⁇ ed and resulted in very accurate transcription. The protocol used is described below.
  • the first PCR amplification mix had 2 ⁇ l of DNA solution; 1 ⁇ l of cassette 7 (primer Cl) ; 1 ⁇ l of cassette 9 (primer (ii) ) ; 10 ⁇ l of 10X LA Ex Taq polymerase buffer; 1 ⁇ l of Ex Taq LA polymerase; 10 ⁇ l of dNTPs (2.5 mM each); and water to a total of 100 ⁇ l.
  • the PCR condition ⁇ were a ⁇ follows: initial dwell 94°C. for 1 min.; amplification (30 cycles): 94°C. for 30 sec; 55°C. for 2 min.; and 72°C. for 1-3 min.; and post dwell at 2°C.
  • a second PCR was conducted under the same conditions except that the genomic DNA solution wa ⁇ replaced by 1 ⁇ l of the first PCR product and cassette 7 (primer Cl) was replaced with cassette 8 (primer C2) .
  • hybridization probes for selecting clones with biotinylated probes is known.
  • a Sigma kit (Cool-1) , Sigma Chemical Co., P.O. Box 14508, St.
  • the beads are then washed 3 times with TE buffer containing 0.1 M NaCl.
  • 100 ⁇ l of genomic DNA that has been pre- denatured at 95°C. for 2 min. is added to the beads.
  • the beads and the DNA are allowed to hybridize far 2 hr. at 42°C. using an equal amount of binding solution that is 2X and consists of 10 mM tris, HCl (pH 7.5), 1 mM EDTA and 2 M NaCl.
  • the temperature is then lowered to room temperature and the beads are washed 3 times in the nybridization solution.
  • the enriched DNA is then eluted by using 0.15 M NaOH containing O.l M NaCL.
  • the DNA is concentrated to 5 ⁇ l in water and cloned by insertion into the pUC18 vector at the Sma I site. The correct pieces are still selected using various biotinylated probes that bind to spider silk DNA sequence ⁇ . Positive clones are sequenced. This technique is very effective but takes quite a bit a work for selection. Enrichment of the DNA can be obtained ⁇ o that only 500 clone ⁇ or le ⁇ need to be ⁇ creened. Without this enrichment, however, 200,000 to 20 million clones must be screened to obtain a clone having the silk gene.
  • the 2 Kb inserts were the longest spider silk pieces cloned. Because of this, it was theorized that a different technique would be required to make larger fragments. It was considered necessary that the technique obtain additional sequence information from parts of the protein coding towards the amino end because, with the available information from the protein sequencing, larger fragments were not produced. Although the 2 Kb piece was over 40% of full length, multimerization was considered necessary to increase strength characteristics -- as strength generally varies with the size of the ⁇ ilk polymer. Therefore, the inventors wanted to multimerize the 2 Kb insert to make a larger protein than the natural gene.
  • PCR would make a suitable method to multimerize these insert ⁇ a ⁇ it avoids the repetition cf reported sequences.
  • the multimerization processes of the present invention are shown in the following examples. Construction of PCR fragments with various u ⁇ eful restriction sites was accomplished by modifying the overhangs of the current beginning and ending primers. Other beginning and ending primers like primers (i) and (ii) described above have different restriction site ⁇ , in addition to having the stop frame codons deleted so that the protein would continue to be translated into mRNA through the sites, enabling longer constructs to be made. The start codons were left in initially so there would be multiple proteins to help check for deletions and to increase the translation.
  • the primers used to make the differing 2 Kb inserts with unique restriction sites are shown below. These are referred to as primers (xxi) - (xxvi) .
  • the ⁇ e band ⁇ were sub ⁇ equently ⁇ eparated by a 1% agaro ⁇ e gel (electrophoresis at 70 V for 90 min. on a 8 cm gel) -- Gene Capsules (Geno Technology, Inc. St. Louis, MO 63108) according to company instruction ⁇ .
  • the band ⁇ were cut with both EcoR I and Hind III re ⁇ triction enzy e ⁇ and the vector 2 ⁇ g wa ⁇ cut with EcoR I and treated with CIAP as described above in Example 2. Then, one half of each of the two 2 Kb pieces and the vector were repurified by phenol extraction and ethanol precipitations and then dissolved into 10 ⁇ l of TE buffer.
  • TE buffer is described in Sambrook et al.
  • the 8 Kb construct of this Example was made exactly as the 6 Kb construct of the above Example with the exception that 4 separate 2 Kb pieces were made from the following four set ⁇ of primer ⁇ : set l: primers (i) and (xxiv); set 2: primers (xxi) and (xxv); set 3: primers (xxii) and (xxvi); and set 4: primers (xxiii) and (ii) .
  • Clones from other techniques such as the cDNA and single site systems described above could also be pieced together to make other multimers.
  • Clones up to 800 kDa are pos ⁇ ible with the multimerization techniques of this invention using full length clones or piece ⁇ therefrom.
  • EX. coli and pUC18 are the preferred initial production systems. Both have good stable expression of high fidelity and excrete the silk protein through their cell membrane. Although only one example of an expression system is given, the specific insert ⁇ coding for natural protein ⁇ or multimer ⁇ derived from them are applicable for use in any vector or genomic incorporation system. Because the potential list of vector ⁇ and hosts is prohibitively long, only a few examples are given below.
  • EX. coli expression systems are preferred because they have the necessary biochemical machinery to produce very high levels of recombinant proteins and excrete them outside the cell membrane. They are also easy to grow using ⁇ imple fermentations. Additionally, many of the major problems for protein production with this system have been overcome as the ⁇ e are among the mo ⁇ t common of expre ⁇ sion systems.
  • pUC18 is among the most commonly used vectors.
  • Other vectors based upon lytic phage, phagamids, and shuttle vectors are also possible as expression insertion systems in addition to the common man-made plasmid ⁇ of which pUC i ⁇ just one.
  • Example ⁇ of such plasmid ⁇ include pBR322, pSP-64, pUR278 and pORFl.
  • Example ⁇ of phage vector ⁇ include lambda, 12001, lambda gtlO, Charon 4a, Charon 40, M13mpl9 and other phage modified from natural bacterial phage.
  • Bacillu ⁇ expre ⁇ sion system ⁇ including Ex. subtilis sy ⁇ terns can also be used. These bacteria have the advantage of good secretion by the host, which results in less processing steps and processing costs. Although an expression cassette might be used, it has been found unneces ⁇ ary with the vector host system ⁇ studied thus far.
  • One phagemid that can act as an EX . coli and Bacillus shuttle vector is pTZ18R which can be obtained from Pharmacia (Piscataway, NJ) .
  • a representative clone has been deposited with the American Type Culture Collection (12301 Parklawn Dr. Rockville, Maryland 20852) on June 2, 1995 and given ATCC No. 69832.
  • the deposit consists of EX . coli XLl MRF' ceils, stiain designation PA21, containing a pUCl ⁇ plasmid (23 Kb) with a full length spider silk gene capable of expressing full length Nephila clavipes silk protein.
  • Saccharomyce ⁇ cerevi ⁇ iae. Schizo ⁇ accharomcyce ⁇ pombe. Pichia pastoris, Asperillus s . , Hansenula s . , and Streptomyces sp. can be used as expres ⁇ ion systems.
  • Aspergillus and Pichia systems there is little evidence that these system ⁇ will produce more protein than bacteria or be amenable to scale-up.
  • These systems might be more desirable to produce USP or food grade materials since bacterial fermentation ⁇ have toxin ⁇ and pyrogen ⁇ associated with them, whereas many of the ⁇ e yea ⁇ t and mold ⁇ y ⁇ tem ⁇ have already been shown to be safe as food grade materials.
  • Plant systems can be used for production of transgenic proteins such as silk. Although the quantity of protein may be les ⁇ than that produced in a microbial system, plant cultivation is rather inexpensive.
  • Agrobacter type transfection system ⁇ that allow genetic incorporation into the plant genome can be used. These may be inserted by bacteria such as Agrobacter tumafaciens LB4404 using gene gun insertion, electroporation or a number of other insertion tools. Once inserted, they can be incorporated into the plant genome in a stable and inheritable manner. These plant systems have a number of benefits, such as being conventionally grown and harvested in large tonnages.
  • Baculovirus expression systems can be used and are well known for high-level expression of recombinant protein ⁇ in insect cell lines. Replication and efficient transfection is accomplished by a number of vectors including pBacPAK6, pBacPAK ⁇ or pBacPAC9. These can be used for high level expres ⁇ ion although they may not be a ⁇ co ⁇ t effective a ⁇ other systems.
  • the fir ⁇ t fermentation ⁇ of tran ⁇ fected ho ⁇ t ⁇ were done in LB broth which consists of 10 grams of bactopeptone, 5 grams of Bacto yeast extract and 5 grams of salt and distilled water to a final concentration of one liter.
  • LB broth which consists of 10 grams of bactopeptone, 5 grams of Bacto yeast extract and 5 grams of salt and distilled water to a final concentration of one liter.
  • either a large amount of precipitate or a cottony mass of spider silk-producing bacteria was observed. This observation was important because it indicated that the proteins were being excreted across the cell membrane. However, these high excretion rates appeared to make the cells somewhat leaky. Therefore, increasing the physiological salt concentration is likely to stabilize the culture.
  • composition of the fermentation media was also found to affect the protease activity. For instance, urea-SDS gels of a two day culture did not show protein degradation when grown in LB broth, but when a culture was grown on LB media supplemented with glucose (10 grams of glucose, 10 grams of peptone and 5 grams of yeast extract and distilled water to one liter) , there was mas ⁇ ive protein degradation after 24 hour ⁇ . The only difference between the ⁇ upplemented LB media and the LB broth wa ⁇ that LB broth contained 10 gm/1 of NaCl, wherea ⁇ the ⁇ upplemented media contained an equivalent amount of glucose.
  • protease inhibitors were inve ⁇ tigated. It was believed that if an inexpensive protease inhibitor could be found and inserted into the culture media, it would be advantageous for fermentation scale-up.
  • the compound ⁇ tested included ZnCl 2 , copper sulfate, disodium EDTA, sodium chloride, boric acid, ethylene glycol bis (B- ax ⁇ notithyi ether) , pnenylmethyl sulfonyl fluoride, N,N,N' ,N' -tetracetic acid, 1,10 phenanthroline, 1,10 phenanthroline iron complex, sucrose, glucose, lactose, fructose, glycerol, peptone and yeast extract.
  • the most effective inhibitors found were salt additions from NaCl or KCl. Boric acid was also found to be a good inhibitor. None of the other compounds were effective. In fact, the simple sugars, and lactose and glucose in particular, promoted protease activity. Peptone and yeast extract did not affect protease activity.
  • the ⁇ e compound ⁇ were te ⁇ ted with AOAC Official Method 969.11, a method for testing proteolytic chillproofing enzymes in beer. To perform this test, 1 ml of the culture was taken and tested. When an active protease was present, the solution cleared in just a few ⁇ econd ⁇ . Protease negative samples showed cloudiness after a ⁇ hr. at 60°C or overnight at 20°C. This test was used as a quick quality control tool to ⁇ creen various culture media for its proteolytic enzyme-inducing ability.
  • Fermentation was attempted using various media. It was found that complex media worked very well. However, acceptable protein production was obtained using 10 times less peptone and yeast extract than contained in LB broth. Thi ⁇ simpler and less expensive media produced considerable protein.
  • This media consisted of the following ingredients: 1.2 g dipotassium phosphate, 1.1 g monosodium phosphate, 4.0 g sodium chloride, 0.45 g magnesium sulfate, 2.0 g ammonium sulfate, 0.04 g sodium nitrate, 0.03 g calcium chloride, 0.02 g ferric sulfate, 0.01 g manganese sulfate, 0.01 g boric acid, 0.0005 g sodium molybdate, 0.005 g cobalt chloride, 0.5 g glycine, 1.0 g alanine, 1.0 g yeast extract, 10 g glycerol, distilled water to 1 liter, pn adjusted to 7.0.
  • a wide range of culture media compositions can be used for the fermentations of this invention. These media can range in composition from salts, glycerol (or other carbon sources) and yeast extract or some other source of minor nutrients. While simpler media is less expensive, it generally result ⁇ in lower level ⁇ of silk protein.
  • the other main fermentation conditions that must be optimized are oxygen, nutrient level and temperature. Anaerobic conditions at 30°C. has been found to be preferred.
  • the carbon source should be added at a relatively high level to maximize growth and protein expres ⁇ ion. For example, 10 gram ⁇ of gluco ⁇ e and 10 gram ⁇ of glycerol per liter ha ⁇ been used.
  • the antibody testing that wa ⁇ developed to determine whether the ⁇ pider silk protein was expres ⁇ ing in the E. coli host was done with three animal ho ⁇ t ⁇ using silkworm ⁇ ilk and ⁇ pider major ampulate gland silk.
  • the silkworm protein was taken from fifth star Bombyx mori caterpillars before they spun a cocoon. By selecting such caterpillars, the silk was viscous and gave the caterpillar a translucent appearance that was recognizable. The viscou ⁇ liquid ⁇ ilk was removed by dis ⁇ ection using aseptic techniques. This ⁇ ilk could then be added to the adjuvant directly. Alternatively, spider silk from the major ampulate gland of the spider could be drawn. However, it was necessary to dissolve the spider silk. Thi ⁇ was done by su ⁇ pending it in 8 M LiBr with heating to 95°C. for 5 min. This spider silk and the silkworm silk were u ⁇ ed for making antibody to the silk.
  • the hemagglutination tests were performed by coating 1% RBCs (Sigma Cat # R-3378) .
  • the dissolved silk was added (lmg) to 1 ml of 1% RBCs. This was vortex mixed a few times at room temperature and refrigerated overnight. The next morning, the RBCs are washed by centrifugation in phosphate buffered saline (pH 7.2) three times to remove any non-adhering protein. The sensitized RBCs were then ⁇ table in the refrigerator for 2 week ⁇ or longer.
  • bacteria cultures (1 ml) were washed 3 times by centrifugation in PBS and brought up into 100 ⁇ l of
  • the present invention also encompasses the techniques for purification and spinning the silk. These steps are essential for the processing of the protein into its final form.
  • the protein can be used as a coating, extruded into a fiber, or made into a polymeric film.
  • the purification of silk protein from the fermentation media can be accomplished by a two step process.
  • the bacterial cells and precipitated protein can be removed by continuous centrifugation.
  • the remaining material present in the fermentation broth can be separated by ultrafiltration since most of the protein above a molecular weight of 80,000 is silk.
  • the protein silk streams from the continuous centrifugation and ultrafiltration procedures can then be combined.
  • the bulk of the remaining proteins can be found in the bacterial membranes.
  • By rupturing the bacterial cells using ultrasound the cells are opened and the ⁇ ilk protein in them i ⁇ removed.
  • Various compounds will keep the silk protein from re- precipitating prior to the spinning process. These include a variety of salts, lithium salts, sodium and pota ⁇ ium hydroxide, urea pho ⁇ phate, guanidine hydrochloride, urea, and hexafluoroi ⁇ opropanol -- all of which dissolve the silk. It was also found by the present inventors that after purification by ultrafiltration, further purification can be effected by alcohol precitation by adding ethanol, methanol, other alcohols or similar solvent ⁇ . This purified silk protein material could be redissolved by ultrasound or by adding one or more of the above salt compound ⁇ .
  • the preferred compounds as determined by cost and environmental consideration ⁇ for ⁇ ilk protein solubilization are sodium and potas ⁇ ium hydroxide, ⁇ odium chloride, potassium chloride and lithium chloride or lithium bromide used in combination with ultrasound or with alcohols for protein purification.
  • spider ⁇ ilk protein is not easily solubilized. Although there is data that ⁇ uggests that ⁇ pider silk may be soluble in harsh chemicals like formic acid (88%) , the present inventors found that it caused degradation of full length protein. However, the present inventor ⁇ found that ⁇ ilk fiber ⁇ could be re ⁇ olubilized in LiSCN, LiBr, LiCl, urea, hexafluoroisopropanol, guanidine hydrochloride and similar denaturants. Once the silk proteins are solubilized, less potent denaturants including urea can be used to prevent the protein from re-precipitating. It most likely will be preferred to use soluble protein before irreversibly spinning into a thread. Therefore, silk protein that has been resolubilized from completely dry silk protein and silk protein that has never been dried completely after being recovered from the fermentation process are recommended for the spinning operations.
  • Silk protein from silkworm ⁇ are typically proce ⁇ sed in the following manner. To make the silk fibers strong enough for weaving, up to five fiber ⁇ are twi ⁇ ted together. After the first reeling, the silk i ⁇ rewound onto ⁇ keins, which are twisted together.
  • the raw silk then goes through several processe ⁇ called throwing.
  • the skeins are washed and dried and wound on large spool ⁇ or bobbin ⁇ .
  • the ⁇ e bobbin ⁇ are placed on doubling frames where single strands are doubled and twisted together to obtain the desired thread size.
  • This thread is then twisted and drawn out by the spindle ⁇ of a throwing frame.
  • the degree of elongation on the throwing frame affects the fiber diameter.
  • the thread On the stretching frame, the thread is made smooth and even.
  • the spider silk proteins produced by the above- de ⁇ cribed method ⁇ can be proce ⁇ ed into fabric ⁇ in the same manner as silkworm proteins. This requires spinning or extruding the protein or protein solutions to obtain silk filaments which may range in diameter from 5 ⁇ m to 200 ⁇ m or higher.
  • the first step in the proce ⁇ is to concentrate the silk proteins from the fermentation solution. This concentration step can be accomplished by a number of methods including the use of membrane technologies which permit only materials of a given molecular weight range to pass. One disadvantage of using these membranes is cost. Other more co ⁇ t effective methods to concentrate the silk proteins and remove the host vector include continuous or batch centrifugation. In addition, ultrasound energy can then be used to lyse the bacterial cell wall and allow the silk proteins produced within the cell wall to escape into the aqueous media. To separate the silk proteins from the bacteria cell walls, higher concentrations of salt ⁇ are favored.
  • the protein solution can be precipitated from media by various alcohols.
  • Useful alcohols include methanol, isopropanol and ethanol.
  • the prior art teaches that at this point in the process the silk proteins can be dissolved in lithium salts and organic solvents containing fluorine. However, that procedure is expensive and a severe environmental challenge.
  • the spider silk proteins are concentrated using alcohols or membrane filters and then maintained in solution in a viscous form by using aqueous solutions of sodium chloride in combination with ultrasound, until they are extruded. If necessary, urea, sodium and potas ⁇ ium hydroxide or lithium salts can be added as di ⁇ closed by prior art processe ⁇ .
  • the protein can be processed in a manner similar to silkworm silk. Once the protein i ⁇ exposed to air and dried, it is no longer soluble in sodium chloride or by ultrasound.
  • the natural colors of the silk protein can be obtained by selecting primers which encode further into the genomic DNA.
  • White, yellow, pink and light purple colors have been ob ⁇ erved with the ⁇ pider ⁇ ilk proteins produced from the clones and processes of the present invention.
  • the selection for natural color is of value for the manufacture of woven textile fabrics since in many cases it will eliminate the need and associated cost of color dying.
  • the spider silk protein filament ⁇ can be treated in a manner ⁇ imilar to silkworm silk ⁇ by winding or twi ⁇ ting two or more thread ⁇ together to make larger yarn ⁇ .
  • the ⁇ e yarn ⁇ can be interwoven with carbon or graphite fibers, boron or boron coated graphite fibers, or Kevlar * to make woven materials of unusually high strength for body armor and other applications.
  • the high strength properties of the spider silk protein filaments permit other processing variables.
  • One such process variable is the on line coating of the silk threads u ⁇ ing variou ⁇ materials to impart color, increased strength, luster, iridescence and other qualities which increase the marketability of the fabric on the basis of appearance, feel or strength.
  • the on line coating can be accomplished by several methods including running the spider silk filaments through various baths or troughs during the extrusion, rewinding or throwing steps. On line vapor deposition can also be used. On line vapor depo ⁇ ition of aterial ⁇ onto silk proteins must take into consideration that some residual salts or other fermentation compounds may be present when the filament is initially formed.
  • these filaments tend to remain wet after being formed unless dried by ovens, fan ⁇ or other mean ⁇ .
  • boiling after extru ⁇ ion may be preferred to remove all trace ⁇ of the fermentation media and resolubilization chemicals -- both of which may invoke an allergenic respon ⁇ e from the skin when woven into fabrics.
  • Material ⁇ that can be vapor depo ⁇ ited onto spider ⁇ ilk proteins include the oxides of tin and titanium. These oxides form a layer on the filaments, the thickness of which depends on the oven condition ⁇ .
  • titanium coating ⁇ may produce higher strength fibers, some people have allergic reactions to titanium dioxide coatings and this may limit it ⁇ u ⁇ e to applications other than clothing.
  • Tin oxides are GRAS (Generally Recommended As Safe) for human skin contact and therefore can be used in clothing applications.
  • Films of spider silk protein can be manufactured by several methods including casting wherein the silk protein solution is poured and spread onto sheets or by using rollers. Films may also be modified by the addition of compounds to the protein prior to casting or rolling. This would include the incorporation of active molecules which may act as fragrances, flavors, absorbents or reactants to various biological reagents and weapon ⁇ . Film ⁇ may also have colors added during pio;eusing or a natural color from a silk clone protein can be selected to impart a natural color.

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Abstract

L'invention concerne de procédés de production de fragments d'ADN codant des protéines de soie provenant d'araignées. Elle concerne également les séquences d'ADN codant lesdites protéines, ainsi que des procédés de production de protéines de soie d'araignée au moyen des séquences d'ADN mentionnées ci-dessus. Ces procédés de clonage et de production de protéines peuvent être appliqués à toutes les araignées productrices de soie. Les clones obtenus au moyen de ces procédés produisent des quantités exploitables commercialement de protéines de soie à poids moléculaire élevé. Etant donné que la soie produite à partir de ces protéines présente des propriétés supérieures de résistance, ces protéines de soie clonées sont d'une importance considérable sur le plan industriel.
PCT/US1996/013767 1995-08-22 1996-08-22 Procedes de clonage servant a obtenir des proteines de soie d'araignee extremement resistantes WO1997008315A1 (fr)

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AU71529/96A AU7152996A (en) 1995-08-22 1996-08-22 Cloning methods for high strength spider silk proteins
JP9510520A JPH11511325A (ja) 1995-08-22 1996-08-22 高力クモシルク蛋白質のクローニング方法
IL12339896A IL123398A0 (en) 1995-08-22 1996-08-22 Cloning methods for high strength spider silk proteins
EP96932937A EP0848754A1 (fr) 1995-08-22 1996-08-22 Procedes de clonage servant a obtenir des proteines de soie d'araignee extremement resistantes
BR9612625A BR9612625A (pt) 1995-08-22 1996-08-22 Processos de clonagem para proteínas de séda de aranha de alta resisténcia

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IL123398A0 (en) 1998-09-24

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