Renaturation of human proinsulin--a study on refolding and conversion to insulin.
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 His(8)-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 (3M guanidinium-HCl or 4M urea) and protein concentrations of 0.5mg/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.
Winter
Jeannette
J
Martin-Luther-Universität Halle-Wittenberg, Institut für Biotechnologie, Kurt-Mothes-Strasse 3, 06120, Halle, Germany.
Lilie
Hauke
H
Rudolph
Rainer
R
D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011384:Proinsulin / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D007328:Insulin; D011994:Recombinant Proteins; D003553:Cystine; D014508:Urea; D011384:Proinsulin; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin; D019791:Guanidine; D003545:Cysteine