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Renaturation of human proinsulin—a study on refolding and conversion to insulin

2002; Elsevier BV; Volume: 310; Issue: 2 Linguagem: Inglês

10.1016/s0003-2697(02)00287-7

1096-0309

Jeannette Winter, Hauke Lilie, Rainer Rudolph,

Transgenic Plants and Applications

The production of human proinsulin in Escherichia coli usually leads to the formation of inclusion bodies. As a consequence, the recombinant protein must be isolated, refolded under suitable redox conditions, and enzymatically converted to the biologically active insulin. In this study we describe a detailed in vitro renaturation protocol for human proinsulin that includes native structure formation and the enzymatic conversion to mature insulin. We used a His8–Arg–proinsulin that was renatured from the completely reduced and denatured state in the presence of a cysteine/cystine redox couple. The refolding process was completed after 10–30 min and was shown to be strongly dependent on the redox potential and the pH value, but not on the temperature. Refolding yields of 60–70% could be obtained even at high concentrations of denaturant (3 M guanidinium–HCl or 4 M urea) and protein concentrations of 0.5 mg/ml. By stepwise renaturation a concentration of about 6 mg/ml of native proinsulin was achieved. The refolded proinsulin was correctly disulfide-bonded and native and monomeric as shown by RP-HPLC, ELISA, circular dichroism, and analytical gel filtration. Treatment of the renatured proinsulin with trypsin and carboxypeptidase B yielded mature insulin.