Patent ID: 6303340
Filing Date: 2001-10-16
Classification: C07K,C12N,C12P,C12Y

Abstract:
A method for producing a purified peptide having a pI below 5 or above 8 which comprises:(a) expressing said peptide having a pI below 5 or above 8, in a recombinant host cell, as a fusion protein in which the peptide is fused at its N-terminus, to a fusion partner comprising an amino acid sequence having an Asp residue at its C-terminus, wherein (i) the C-terminal Asp residue of the fusion partner and the N-terminal residue of the peptide form a bond which is cleavable under acidic conditions, (ii) the peptide has a different net charge from that of the fusion partner and any undesired fragments produced by the acid cleavage of the fusion partner to allow it to be separated from the fusion partner and any undesired acid cleavage fragments of the fusion protein by ion-exchange chromatography and (iii) the fusion protein forms inclusion bodies within the recombinant host cell;(b) recovering the inclusion bodies from the recombinant host cell;(c) cleaving the peptide from the fusion partner by subjecting the fusion protein to acidic conditions in the absence of a chaotrope;(d) solubilizing insoluble cleavage products by treating them with 3.5 M urea, so that the primary structure of the peptide is not degraded; and(e) isolating the peptide by ion-exchange chromatography.