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

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.

Keywords:

• High performance hydrophobic interaction chromatography
• Protein refolding
• Purification
• Recombinant human proinsulin

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 29675017 and 39880003), the Foundation of the Young Teacher in the University of Ministry of Education P. R. C (Grant No. DF00308), the Natural Science Foundation in Shaanxi Province (Grant No. 2001H02), and the Foundation of the Committee of Education in Shaanxi Province (Grant No. 99JK101). The inclusion bodies of rh‐proinsulin, expressed in E. coli and standard rh‐proinsulin, were provided by Xuzhou Biochemical Pharmaceutical Plant, Jiangsu Province, China.