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1 Department of Bioengineering, Xi’an Jiaotong University, Xi’an 710049, China
2 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, Shanghai 200031, China
Reprint requests to: You-Min Feng, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China; e-mail: fengym{at}sunm.shcnc.ac.cn; fax: (86) 021-64338357.
Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20–B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20-B19]PIP) containing the single disulfide A20–B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2–3 magnitudes compared with that of the wild-type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20–B19, which suggested that formation of the disulfide A20–B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20–B19.
Keywords: Insulin; folding; disulfide; kinetics; thermodynamics
Abbreviations: PIP, a recombinant single-chain insulin in which the C terminus of porcine insulin B-chain and the N terminus of porcine insulin A-chain were linked together by a dipeptide, Ala-Lys • IGF-1, insulin-like growth factor 1 • BPTI, bovine pancreatic trypsin inhibitor • RNaseA, ribonuclease A • EGF, epidermal growth factor • GSH, reduced glutathione • GSSG, oxidized glutathione • EDTA, ethylenediaminetetraacetic acid • HPLC, high performance liquid chromatography • TFA, trifluoroacetic acid • PAGE, polyacrylamide gel electrophoresis • UV, ultraviolet • CD, circular dichroism • NMR, nuclear magnetic resonance
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