Abstract
A method for the simplified, reproducible production of both normal and altered versions of human proinsulin has been developed. A polyhistidine/proinsulin fusion protein was expressed using a prokaryotic expression system and partially purified by affinity chromatography. Disulfide bonds within the polypeptide were formed prior to removal of the affinity tag. The proinsulin cleaved from the fusion protein was then subjected to a final purification step of semipreparative reversed-phase high-performance liquid chromatography. Integrity of both the normal and mutant proinsulins was confirmed by peptide mapping and mass spectrometry. The different versions of proinsulin will be used to map those residues of the substrate used in cleavage site recognition by members of the furin/PC family of converting enzymes.