Human insulin production from a novel mini-proinsulin which has high receptor-binding activity

Abstract

To increase the folding efficiency of the insulin precursor and the production yield of insulin, we have designed a mini-proinsulin (M2PI) having 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 His10 tag for affinity purification. The gene for the fusion protein was inserted downstream of the T7 promoter of the expression plasmid pET-3a, and the fusion proteins were produced as inclusion bodies in the Escherichia coli cytoplasm at levels 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, indicating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide. Native human insulin was successfully generated by subsequent enzymic conversion of mini-proinsulin. 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 folding and activity of proinsulin.

Biological treatment of toluene, acetone, and methyl ethyl ketone vapors with a biofilter packed with glass packing materials

“…The biofilm systems were operated at 24-28 • C at an empty bed retention time (EBRT) of 15-60 s under nitrogen deficient conditions (0.06 N g/d) where nutrient solution was supplied only 1 L every 2 weeks. MPI is a proinsulin having only nine amino acid residues in the C peptide chain (RRYPGNVKR) (Shin et al, 1997;Chang et al, 1998). 27.5 g/m 3 /h) of toluene, hydrophobic aromatic compound, was obtained at a loading rate of 3.7-62.5 g/m 3 /h.…”

Glucagon-induced self-association of recombinant proteins inEscherichia coli and affinity purification using a fragment of glucagon receptor

“…1). The T S and T L peptides, derived from N-terminal human TNF-␣ (known as forming ␤-sheet structure) were previously reported as effective fusion partners in the high-level synthesis of other therapeutic proteins (Chang et al, 1998;Shin et al, 1997;1998) and also employed in this study to clarify the role of glucagon fusion. After gene expression, all recombinant fusion proteins were accumulated in the form of insoluble aggregates (inclusion bodies) in bacterial cytoplasm, and all isolated aggregates involving recombinant fusion proteins were easily dissolved at room temperature by simple pH shift (→12 →8) only without using any denaturing agents.…”

Bioorganic synthesis of a recombinant HIV-1 fusion inhibitor, SC35EK, with an N-terminal pyroglutamate capping group

“…Such proteins include antibiotic peptides, 11 zinc finger peptides, 12 insulin-like peptides 13 and pH-responsive self-assembling peptides. 14 It is noteworthy that CNBr-mediated cleavage releases the first fragment containing a cyclic homoserine lactone (Hsl) at the C-terminus, 15 and the second fragment without any N-terminal functional group.…”