<Emphasis Type="Italic">In vitro</Emphasis> insulin refolding: Characterization of the intermediates and the putative folding pathway |
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Authors: | Tang YueHua Wang Shuai Chen Yan Xu GenJun Feng YouMin |
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Institution: | 1.Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai,China;2.College of Life Science,Zhejiang Sci-Tech University,Hangzhou,China |
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Abstract: | 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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Keywords: | insulin insulin refolding folding intermediate folding pathway disulfide |
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