Synthesis of spider dragline and/or flagelliform proteins
The present invention is directed to a method of producing spider dragline and/or flagelliform proteins. The invention is further directed to a method of producing spider threads and to a dragline/flagelliform protein or dragline/flagelliform protein thread produced by these methods. The invention further provides the use of these proteins/threads in the field of biotechnology and/or medicine, in particular in the manufacture of wound closure or coverage systems, suture materials and in the manufacture of replacement materials, preferably artificial cartilage or tendon materials.
1. A method of producing spider dragline proteins comprising the following steps:
a) providing a nucleic acid sequence coding for an Araneus diadematus fiber 4 (ADF-4) protein encoded by SEQ ID NO:2 or a variant thereof that differs from the protein encoded by SEQ ID NO:2 by no more than one amino acid substitution, no more than five amino acid deletions, no more than five amino acid insertions, and no more than twenty amino acid additions,
b) introducing the nucleic acid sequence(s) provided in a) into an insect cell,
c) expressing the proteins; and
d) recovering said proteins.
2. The method of claim 1 , further comprising providing in step a), a second nucleic acid sequence, coding for an Araneus diadematus fiber 3 (ADF-3) protein encoded by SEQ ID NO: 1or a variant thereof that differs from the protein encoded by SEQ ID NO:1 by no more than one amino acid substitution, no more than five amino acid deletions, no more than five amino acid insertions, and no more than twenty amino acid additions.
3. The method of claim 1 , wherein the nucleic acid sequence is contained in a vector.
4. The method of claim 3 , wherein the vector is an expression vector, which comprises one or more regulatory sequences.
5. The method of claim 4 , wherein the regulatory sequences comprise promoter p10 and/or polyhedrin.
6. The method of claim 3 , wherein the vector is a viral vector.
7. The method of claim 6 , wherein the viral vector is selected from the group consisting of a baculovirus vector system and a vaccinia virus vector system.
8. The method of claim 1 , wherein the insect cell is from a lepidopteran insect.
9. The method of claim 8 , wherein the insect cell is a Sf9, Sf21 or Trichoplusia ni cell.
10. The method of claim 8 , wherein the lepidopteran insect is selected from the group consisting of Spodoptera frugiperda and Trichoplusia ni.
11. The method of claim 1 , wherein the only dragline protein expressed is ADF-4 encoded by SEQ ID NO: 2.
12. The method of claim 1 , wherein the expression of said protein occurs by secretory or cytoplasmatical expression.
13. A method for producing threads from spider dragline proteins, comprising the following steps: a) expressing the ADF-4 proteins encoded by SEQ ID NO:2 or a variant thereof that differs from the protein encoded by SEQ ID NO:2 by no more than one amino acid substitution, no more than five amino acid deletions, no more than five amino acid insertions, and no more than twenty amino acid additions, b) recovering said proteins, and c) spinning said proteins into threads by a suitable method.
14. The method of claim 13 , wherein the only dragline protein is ADF-4 encoded by SEQ ID NO: 2.
15. The method of claim 13 , further comprising providing a second nucleic acid sequence, coding for an ADF-3 protein encoded by SEQ ID NO: 1 or a variant thereof that differs from the protein encoded by SEQ ID NO:1 by no more than one amino acid substitution, no more than five amino acid deletions, no more than one amino acid insertions, and no more than twenty amino acid additions, and expressing said ADF-3 protein in step a).
16. A vector, which comprises a nucleic acid sequence coding for an ADF-4 protein encoded by SEQ ID NO:2 or a variant thereof that differs from the protein encoded by SEQ ID NO:2 by no more than one amino acid substitution, no more than five amino acid deletions, no more than five amino acid insertions, and no more than twenty amino acid additions.
17. A baculovirus vector, which comprises a nucleic acid sequence coding for an ADF-4 protein encoded by SEQ ID NO:2 or a variant thereof that differs from the protein encoded by SEQ ID NO:2 by no more than one amino acid substitution, no more than five amino acid deletions, no more than five amino acid insertions, and no more than twenty amino acid additions.