PRODUCTION OF RECOMBINANT PROTEIN BY AUTOPROTEOLYTIC CLEAVAGE OF FUSION PROTEIN

摘要

PROBLEM TO BE SOLVED: To provide a method for recombinant production of a heterologous polypeptide of interest, comprising, (i) cultivation of a host cell which is transformed with an expression vector which comprises a nucleic acid molecule which encodes a fusion polypeptide, the fusion polypeptide comprising a derivative of an autoprotease Nproof Pestivirus, wherein at least one cysteine residue of the naturally occuring autoprotease Nproof Pestivirus is replaced by another amino acid residue, and a second polypeptide which is connected to the first polypeptide at the C-terminus of the first polypeptide in a manner such that the second polypeptide is capable of being cleaved from the fusion polypeptide by the autoproteolytic activity of the first polypeptide, the second polypeptide being a heterologous polypeptide, wherein cultivation occurs under conditions which cause expression of the fusion polypeptide and formation of corresponding cytoplasmic inclusion bodies, (ii) isolation of the inclusion bodies from the host cell, (iii) solubilization of the isolated inclusion bodies, (iv) induction of autoproteolytic cleavage of the heterologous polypeptide of interest from the fusion polypeptide, and (v) isolation of the cleaved heterologous polypeptide of interest.

SOLUTION: A certain derivative of the naturally occuring autoprotease Nproof Pestivirus having low pI and increased solubility is found to be suitable for use under wide conditions.

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