Abstract
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 8.0 mol 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 the rh‐proinsulin. Thus, the technology for the renatured and purified rh‐proinsulin is very simple and fast.
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