EP0848754A1 - Klonierungsverfahren für hochfeste spinnenseideproteine - Google Patents

Klonierungsverfahren für hochfeste spinnenseideproteine

Info

Publication number
EP0848754A1
EP0848754A1 EP96932937A EP96932937A EP0848754A1 EP 0848754 A1 EP0848754 A1 EP 0848754A1 EP 96932937 A EP96932937 A EP 96932937A EP 96932937 A EP96932937 A EP 96932937A EP 0848754 A1 EP0848754 A1 EP 0848754A1
Authority
EP
European Patent Office
Prior art keywords
dna
silk
protein
spider
primers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96932937A
Other languages
English (en)
French (fr)
Inventor
Richard M. Basel
Glenn R. Elion
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0848754A1 publication Critical patent/EP0848754A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
EP96932937A 1995-08-22 1996-08-22 Klonierungsverfahren für hochfeste spinnenseideproteine Withdrawn EP0848754A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US51769495A 1995-08-22 1995-08-22
US517694 1995-08-22
PCT/US1996/013767 WO1997008315A1 (en) 1995-08-22 1996-08-22 Cloning methods for high strength spider silk proteins

Publications (1)

Publication Number Publication Date
EP0848754A1 true EP0848754A1 (de) 1998-06-24

Family

ID=24060849

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96932937A Withdrawn EP0848754A1 (de) 1995-08-22 1996-08-22 Klonierungsverfahren für hochfeste spinnenseideproteine

Country Status (7)

Country Link
EP (1) EP0848754A1 (de)
JP (1) JPH11511325A (de)
CN (1) CN1200145A (de)
AU (1) AU7152996A (de)
BR (1) BR9612625A (de)
IL (1) IL123398A0 (de)
WO (1) WO1997008315A1 (de)

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CA2351274C (en) * 2000-06-29 2011-04-26 Fiona F. Hunter Isolated cocoon silk protein from simulium vittatum and nucleic acids encoding such protein
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Also Published As

Publication number Publication date
AU7152996A (en) 1997-03-19
WO1997008315A1 (en) 1997-03-06
BR9612625A (pt) 1999-06-01
CN1200145A (zh) 1998-11-25
IL123398A0 (en) 1998-09-24
JPH11511325A (ja) 1999-10-05

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