US Patent 7829319 Glutamic acid-modified classical swine fever virus autoproteases N

The invention relates to a process for the recombinant production of a heterologous polypeptide of interest by cultivating a bacterial host cell transformed with an expression vector comprising a nucleic acid molecule encoding a fusion polypeptide wherein (a) the amino-proximal fusion partner is an autoprotease Npro comprising the replacement(s) by glutamic acid of one or more cysteines at positions corresponding to the positions 112, 134, and 138 of the autoprotease Npro of classical swine fever virus and (b) the carboxyl-proximal fusion partner is an heterologous polypeptide of interest fused to the autoprotease Npro so that it is capable of being cleaved from the fusion polypeptide by autoprotease Npro autoproteolytic activity, said process comprising (i) cultivating the transformed host cell under conditions permitting the expression of the fusion polypeptide and the 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 cleaved heterologous polypeptide of interest.