Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli

Michael D. Jones, Linda O. Narhi, Wen Chang Chang, Hsieng S. Lu

研究成果: 雜誌貢獻文章

13 引文 斯高帕斯(Scopus)

摘要

Oxidative folding of recombinant human stern cell factor (rhSCF) produced in Escherichia coli was investigated in vitro. Folding of denatured and reduced rhSCF involves at least five intermediate forms, I-1 to I-5, detectable by their differences in hydrophobicity using reverse-phase high performance liquid chromatography. Both I-1 and I-2 contain a native-like disulfide bond, Cys4-Cys89 and Cys43-Cys138, respectively, and I-3 forms a mispaired disulfide, Cys43-Cys89. These forms appear to reach steady state equilibrium and are important folding intermediates. I-1 was found to be the prominent intermediate that directly folds into native rhSCF (N); and the thermodynamically less stable I-2 favors rearrangment into I-1. I-3 may serve as an intermediate for disulfide rearrangement between I-1 and 1-2. I-4 and I-5, which are disulfide-linked dimers, are in equilibrium with reduced rhSCF and other intermediates and may not play an important role in rhSCF folding. Both trifluoroacetic acid-trapped I-1 and I-2, after isolation by high performance liquid chromatography, proceed with the remaining oxidative folding process after reconstitution. Iodoacetate-trapped I-1 and I-2 contain low α-helical content and some tertiary structure, while I-3 and reduced rhSCF have little ordered structure. Gel filtration/light-scattering experiments indicate that reduced rhSCF and iodoacetate-trapped I-1, I-2, and I-3 exist as dimeric forms, indicating that rhSCF dimerization precedes formation of disulfide bonds. I-1, I-2, I-3, and the C43,138A analog lacking Cys43-Cys138 bond are not biologically active or exhibit significantly lower activity. The two disulfide bonds in rhSCF seem to be essential for the molecule to maintain an active conformation required for its receptor binding and biological activities.
原文英語
頁(從 - 到)11301-11308
頁數8
期刊Journal of Biological Chemistry
271
發行號19
DOIs
出版狀態已發佈 - 六月 4 1996
對外發佈Yes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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