Citation: Cui E.Lin, Quan Bai. Short communication Refolding of reduced/denatured bovine pancreatic insulin with ion-exchange chromatography coupled with MALDI-TOF MS[J]. Chinese Chemical Letters, ;2009, 20(12): 1487-1490. doi: 10.1016/j.cclet.2009.07.005 shu

Short communication Refolding of reduced/denatured bovine pancreatic insulin with ion-exchange chromatography coupled with MALDI-TOF MS

Cui E.Lin ,
Quan Bai

Institute of Modern Separation Science, Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Northwest University, Xi'an 710069, China

Received Date: 26 March 2009

Fund Project: This work is supported by the National 863 Program (No.2006AA02Z227)the Foundation of Key Subject Construct of Analytical Chemistry in Shaanxi Province and the Foundation of Key Laboratory of Modern Separation Science in Shaanxi Province (No.05JS62).

The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anionexchange chromatography (WAX) coupled with MALDI-TOF MS.The results indicated that the disulfide bonds almost cannot be formed correctly with the common mobile phase by WAX.However, with the urea gradient elution and in the presence of GSSG/ Cyst as the ratio 1:6 in the mobile phase employed, the disulfide exchange of reduced/denatured insulin can be accelerated resulting in forming the correct three disulfide bonds.The protein refolding efficiency of reduced/denatured insulin can be increased from 3% to 34%.The effects of urea gradient and the oxidant and reductant groups, such as GSSG/GSH, Cyst, and GSSG/Cyst, on the forming the disulfide bonds of reduced/denatured insulin were investigated in detail.The results were further tested by the separation of the WAX fraction of reduced/denatured insulin with RPLC and MALDI-TOF MS.
- Protein refolding,
- Liquid chromatography,
- Reduced/denaturation,
- Insulin,
- MALDI-TOF MS
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