Studies on Renaturation with Simultaneous Purification of Recombinant Human Proinsulin from E. coli with High Performance Hydrophobic Interaction Chromatography

摘要

The renaturation with simultaneous purification of recombinant human proinsulin (rh-proinsulin) expressed in E. coli by high performance hydrophobic interaction chromatography (HPHIC) was investigated. The result indicates that the reduced/denatured rh-proinsulin, extracted with 8.0mol L~(-1) urea solution in the presence of β-mercaptoethanol can be renatured and purified, simultaneously, in 45 min with HPHIC, resulting in the purity and mass recovery being more than 90% and 94%, respectively. The disulfide bonds of rh-proinsulin can correctly form on the HPHIC column without the presence of reduced and oxidized glutathione (GSH, GSSG). The renaturation efficiency of rh-proinsulin with HPHIC was tested by enzyme cleavage in order to obtain insulin. The result was also confirmed with RPLC, SDS-PAGE, and MALDI-TOF, respectively. The renatured and purified rh-proinsulin can directly be enzyme-cleaved in the collected fraction containing rh-proinsulin. Thus, the technology for the renatured and purified rh-proinsulin is very simple and fast.