Designing structural-motifs for the preparation of acylated proinsulin and their regiospecific conversion into insulin modified at Lys29 (K29)

Abstract

Eight proinsulin encoding genes were prepared and their translation products, when treated with a cocktail of trypsin and carboxypeptidase B, analyzed for the following features. One, their ability to undergo facile removal of the N-terminal linker, generating the phenylalanine residue destined to be the N-terminal of the B-chain of insulin, at a rate similar to that involved in the removal of the C-peptide. Two, processing of diarginyl insulin, produced in the latter process, by carboxypeptidase B then needed to be rapid to remove the two arginine residues, Three, both these operations were to be efficient whether the N-terminal methionine was acylated or not. Four, the proinsulin constructs needed to contain a minimum number of sites for acylation. The aforementioned features were monitored by mass spectrometry and the proinsulin derivative containing MRR at the N-terminal and K64 mutated to Q64, designated as MRR-(Q64) human proinsulin [MRR-(Q64) hpi] optimally fulfilled these requirements. The derivative was smoothly acylated with reagents of two chain lengths (acetyl and dodecanoyl) to give acetyl/dodecanoyl MRR-(Q64) hpi. Acetyl MRR-(Q64) hpi, using the cocktail of the two enzymes, was smoothly converted into, acetyl insulin. However, when dodecanoyl MRR-(Q64) hpi was processed with the above cocktail, carboxypeptidase B (whether from animal pancreas or recombinant) showed an unexpected specificity of acting on the K29-T30 bond of the insulin derivatives when K29 contained a large hydrophobic acyl group, generating dodecanoyl des-30 insulin.