Modified spider silk proteins

1 . A method of modifying a spider silk protein comprising the steps of:

a) providing a nucleic acid encoding a spider silk protein or a fragment or variant thereof not containing lysine or cysteine residues;

b) replacing nucleic acids encoding one or more amino acids in said spider silk protein by a lysine or cysteine encoding nucleic acid sequence, or adding a nucleic acid sequence containing nucleic acids encoding lysine and/or cysteine to said sequence;

c) expressing the modified nucleic acid sequence obtained in b) in a suitable host, and

d) recovering the expressed modified spider silk protein.

2 . The method of claim 1 , which further comprises coupling other substances to said lysine and/or cysteine molecules in the modified spider silk protein.

3 . The method of claim 1 or 2 , wherein the one or more amino acids replaced in step b) are selected from the group consisting of glycine, alanine, serine, glutamate, aspartate and threonine.

4 . The method of claim 1 , wherein the substance to be coupled to lysine and/or serine is selected from the group consisting of polypeptides, polysaccharides, marker molecules, quantum dots, metals, nucleic acids, lipids and low molecular drugs.

5 . The method of claim 4 , wherein the low molecular drugs are selected from drugs containing a carboxyl, carbonyl, imido or thiol group.

6 . The method of claim 1 , wherein the spider silk protein provided in a) is based on a dragline and/or flagelliform protein.

7 . The method of claim 6 , wherein the dragline and/or flagelliform protein provided in a) is selected from a dragline or flagelliform protein of orb-web spiders (Araneidae and Araneoids).

8 . The method of claim 7 , wherein the dragline proteins and/or flagelliform proteins provided in a) are derived from Araneus diadematus and/or Nephila clavipes.

9 . The method of claim 8 , wherein the fragment is a module, wherein the module comprises one or more polyalanine containing consensus sequences.

10 . The method of claim 9 , wherein the polyalanine containing consensus sequence is derived from ADF-3 and has the amino acid sequence of SEQ ID NO: 1 (module A) or a variant thereof.

11 . The method of claim 8 , wherein the fragment is a module derived from ADF-3 and comprises the amino acid sequence of SEQ ID NO: 2 (module Q) or a variant thereof.

12 . The method of claim 10 and 11 , wherein the spider silk protein provided in step a) comprises one or more of (AQ) and/or (QAQ).

13 . The method of claim 12 , wherein the spider silk protein comprises (AQ) 12 , (AQ) 24 , (QAQ) 8 or (QAQ) 16 .

14 . The method of claim 8 , wherein the fragment is a module derived from ADF-4 comprising the amino acid sequence of SEQ ID NO: 3 (module C) or a variant thereof.

15 . The method of claim 14 , wherein the spider silk protein provided in a) comprises C 16 or C 32 .

16 . The method of claim 8 , wherein the fragment is a module derived from a flagelliform protein and is module K (SEQ ID NO: 4), module sp (SEQ ID NO: 5), module X (SEQ ID NO: 6), and/or module Y (SEQ ID NO: 7) or a variant thereof.

17 . The method of claim 16 , wherein the spider silk protein comprises Y 8 , Y 16 , X 8 , X 16 , K 8 , K 16 or Y 6 X 2 sp 1 K 2 Y 2 .

18 . The method of claim 1 , wherein the modified spider silk protein recovered in step d) comprises one or more of the modules of SEQ ID NO: 8-19.

19 . The method of claim 1 , wherein to the modified spider silk protein recovered in step d) or to the spider silk protein provided in step a) a nucleic acid encoding an amino terminal TAG according to SEQ ID NO: 20-24 and/or a carboxyl terminal TAG of SEQ ID NO: 25-28 is added.

20 . A modified spider silk protein obtainable by the method of claim 1 .

21 . A modified spider silk protein comprising one or more of the modules of SEQ ID NO: 8-28.

22 . A nucleic acid sequence coding for a modified spider silk protein as obtained in step d) of claim 1 or for a modified spider silk protein of claims 20 or 21 .

23 . An expression vector, which comprises the nucleic acid sequence of claim 22 and one or more regulatory sequences.

24 . The vector of claim 23 , which is a plasmid or a viral vector, preferably a baculovirus system or a vaccinia virus vector system.

25 . A vector, which comprises the nucleic acid sequence of claim 22 and which preferably is derived from the cloning vector of SEQ ID NO: 29 or a variant thereof.

26 . A host, which has been transformed with the vector of claim 23 .

27 . The host of claim 26 , which is a prokaryotic cell.

28 . The host of claim 27 , which is E. coli or Bacillus subtilis.

29 . The host of claim 26 , which is a eukaryotic cell.

30 . The host of claim 29 , which is a mammalian cell, plant cell, yeast cell or an insect cell.

31 . The mammalian cell of claim 30 , which is a CHO, COS, HeLa, 293T, HEH or BHK cell.

32 . The host of claim 31 , which is a yeast cell.

33 . The host of claim 32 , which is Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Candida albicans, Hansenula polymorpha.

34 . The host of claim 30 , wherein the insect cell is selected from Lepidoptera insect cells, preferably from Spodoptera frugiperda and from Trichoplusia ni.

35 . The host of claim 34 , wherein the insect cell is a Sf9, Sf21 or high five cell.

36 . The host of claim 30 , wherein the plant cell is derived from tobacco, potato, corn, pea and tomato.

37 . A pharmaceutical or cosmetical composition containing a modified spider silk protein of claim 20 or 21 and a pharmaceutically acceptable carrier.

38 . A fibre/thread, filament, film, foam, sphere, nanofibril, hydrogel and the like made from the modified spider silk protein of claim 20 or 21 .

39 . A paper product, comprising a modified spider silk protein of claims 20 or 21 .

40 . A textile or leather product, comprising a recombinant spider silk protein of claims 20 or 21 .

41 . The paper product, textile or leather product of claims 39 or 40 , wherein the recombinant spider silk protein is present as a coating.

42 . A gel or a foam comprising or consisting of a protein of claims 20 or 21 .