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In vitro insulin refolding: characterization of the intermediates and the putative folding pathway
The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all...
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Published in: | Science China. Life sciences 2007-12, Vol.50 (6), p.717-725 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding. |
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ISSN: | 1006-9305 1674-7305 1862-2798 1869-1889 |
DOI: | 10.1007/s11427-007-0092-3 |