INTEGRATED PRODUCTION OF HUMAN INSULIN AND ITS C-PEPTIDE

The potential for the development of an integrated process for product ion of human insulin and its C-peptide in Escherichia coli has been in vestigated. Human proinsulin was produced intracellularly in E, coil f used to two synthetic IgG-binding domains (ZZ) derived from staphyloco ccal protein A. High expression levels (3 g/l culture) of the gene pro duct, which accumulated as inclusion bodies, was obtained. Solubilizat ion of inclusion bodies by oxidative sulfitolysis and subsequent renat uration was performed directly after cell lysis and pellet wash. IgG a ffinity chromatography was used for efficient recovery of pure proinsu lin fusion protein in a single step. Monomers of the proinsulin fusion protein constituted similar to 70%. A single step conversion of the f usion protein into insulin and C-peptide by trypsin and carboxypeptida se B treatment was achieved by engineering the junction between proins ulin and its affinity handle, ZZ. Characterization of the cleavage pro ducts by reversed phase chromatography (RPC) verified that human insul in and C-peptide were generated and that the ZZ affinity handle was re sistant to cleavage. Human insulin and C-peptide were recovered with h igh yields by preparative reversed-phase high performance liquid chrom atography (RP-HPLC). The potential use of the presented scheme for lar ge-scale production of recombinant insulin and/or its C-peptide is dis cussed.