| 摘要 |
The invention relates to a method for producing polypeptides or proteins in a two-chain form by recombinant expression in E. coli host cells, whereby i) the polypeptide or protein exerts its biological activity as a two-chain polypeptide or protein, ii) the C-terminal amino acid group of the first chain is an Arg or Lys group, iii) the second chain of the protein/polypeptide, at its N terminus, has 1 to 20 amino acid groups and a pentapeptide sequence VPXGS referred to as PRS, wherein X may be any naturally occurring amino acid, wherein V represents Val, Leu, Ile, Ala, Phe, Pro or Gly, P represents Pro, Leu, Ile, Ala, Phe, Val or Gly, G represents Gly, Leu, Ile, Ala, Pro, Phe or Val and S represents Ser, Tyr, Trp or Thr; and (iv) the method comprises the following steps: a) modifying the polypeptide or protein on the nucleic acid level in such a manner that the polypeptide or protein in its modified form comprises the sequence VPXGS in its loop area, with X, V, P, G and S being defined as above; b) introducing the construct modified on the nucleic acid level into E. coli cells; c) cultivating and then lysing the host cells; and d) isolating the two-chain polypeptide or protein. According to a preferred embodiment of the invention, the polypeptide or protein is a botulinum neurotoxin, especially the botulinum neurotoxin (BoNT(A)) type A.
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