Production of recombinant proteins by autoproteolytic...

Details Production of recombinant proteins by autoproteolytic... Production of recombinant proteins by autoproteolytic...

C - Chemistry, Metallurgy

12

N

C12N 9/50 (2006.01) C07K 19/00 (2006.01) C12N 15/09 (2006.01) C12N 15/57 (2006.01) C12N 15/62 (2006.01) C12N 15/63 (2006.01) C12P 21/06 (2006.01)

Patent

CA 2605140

The invention relates to a process for the recombinant production of a heterologous polypeptide of interest, comprising, (i) cultivation of a bacterial host cell which is transformed with an expression vector which comprises a nucleic acid molecule which codes for a fusion polypeptide, the fusion polypeptide comprising a derivative of an autoprotease Npro of Pestivirus, wherein at least one cysteine residue of the naturally occuring autoprotease Npro of 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, said 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.

L'invention concerne un procédé de production recombinante d'un polypeptide hétérologue recherché, qui consiste à: (a) mettre en culture une cellule bactérienne hôte transformée par un vecteur d'expression qui contient une molécule d'acide nucléique codant un polypeptide de fusion, le polypeptide de fusion comprenant un dérivé d'une autoprotéase Npro de Pestivirus, au moins un résidu cystéine de l'autoprotéase Npro, d'origine naturelle, de Pestivirus étant remplacé par un autre résidu d'aminoacide, et un second polypeptide lié au premier polypeptide par l'extrémité C-terminal de celui-ci de manière à ce que le second polypeptide puisse être coupé du polypeptide de fusion sous l'effet de l'activité autoprotéolytique du premier polypeptide, le second polypeptide étant un polypeptide hétérologue; la culture se produit en outre dans des conditions qui déclenchent l'expression du polypeptide de fusion ainsi que la formation de corps d'inclusions cytoplasmiques correspondants; (b) isoler les corps d'inclusions de la cellule hôte; (c) solubiliser les corps d'inclusions isolés; (d) déclencher le clivage autoprotéolytique du polypeptide hétérologue recherché du polypeptide de fusion; et enfin, (e) isoler le polypeptide hétérologue recherché ainsi coupé.

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