HUMAN INSULIN PRODUCTION FROM A NOVEL MINI-PROINSULIN WHICH HAS HIGH RECEPTOR-BINDING ACTIVITY

To increase the folding efficiency of the insulin precursor and the pr oduction yield of insulin, we have designed a mini-proinsulin (M2PI) h aving the central C-peptide region replaced with a sequence forming a reverse turn. The mini-proinsulin was fused at the N-terminus to a 21- residue fusion partner containing a His(10) tag for affinity purificat ion. The gene for the fusion protein was inserted downstream of the T7 promoter of the expression plasmid pET-3a, and the fusion proteins we re produced as inclusion bodies in the Escherichia coli cytoplasm at l evels up to 25% of the total cell protein. The protein was sulphonated , cleaved by CNBr and the M2PI mini-proinsulin was purified using ion- exchange chromatography. The refolding yield of M2PI was 20-40% better than that of proinsulin studied at the same molar concentrations, ind icating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide. Native human insulin was succesfully generated by subsequent enzymic conversion of mini-pr oinsulin. The mini-proinsulin exhibited high receptor-binding activity , about 50% as potent as insulin, suggesting that this single-chained mini-proinsulin may provide a foundation in understanding the receptor -bound structure of insulin as well as the role of C-peptide in the fo lding and activity of proinsulin.