PRODUCTION OF RECOMBINANT PROTEINS BY AUTOPROTEOLYTIC CLEAVAGE OF A FUSION PROTEIN

摘要

PROBLEM TO BE SOLVED: To provide a method for recombination production of a heterologous polypeptide of interest comprising the steps of: (i) culturing a host cell transformed with an expression vector comprising a nucleic acid molecule encoding a fusion polypeptide, where the fusion polypeptide comprises a first polypeptide being a derivative of wild type autoprotease Nof Pestivirus having at least one cysteine residue of the wild type autoprotease replaced by another amino acid residue, and a second polypeptide being a heterologous polypeptide linked to the C terminus of the first polypeptide in such a manner that the second polypeptide can be cleaved from the first polypeptide by the autoproteolytic action of the first polypeptide, and the culture is carried out under the conditions enabling expression of the fusion polypeptide and formation of corresponding cytoplasmic inclusion bodies; (ii) isolating the inclusion bodies from the host cell; (iii) solubilizing the isolated inclusion bodies; (iv) inducing autoproteolytic cleavage of the heterologous polypeptide of interest from the fusion polypeptide; and (v) isolating the heterologous polypeptide of interest.SOLUTION: The invention is based on the finding that certain derivatives of wild type pestiviral autoprotease N, having a low pI and high solubility, are suitable for use under broad conditions.