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

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)11301-11308
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number19
DOIs
Publication statusPublished - Jun 4 1996
Externally publishedYes

Fingerprint

Stem Cell Factor
Disulfides
Escherichia coli
Oxidation
Iodoacetates
High performance liquid chromatography
Trifluoroacetic Acid
Dimerization
Hydrophobicity
Bioactivity
High Pressure Liquid Chromatography
Dimers
Light scattering
Conformations
Gels
Reverse-Phase Chromatography
Hydrophobic and Hydrophilic Interactions
Molecules
Gel Chromatography
Light

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli. / Jones, Michael D.; Narhi, Linda O.; Chang, Wen Chang; Lu, Hsieng S.

In: Journal of Biological Chemistry, Vol. 271, No. 19, 04.06.1996, p. 11301-11308.

Research output: Contribution to journalArticle

Jones, Michael D. ; Narhi, Linda O. ; Chang, Wen Chang ; Lu, Hsieng S. / Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 19. pp. 11301-11308.
@article{484571cec73d42218e2d43d6e2b89579,
title = "Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli",
abstract = "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.",
author = "Jones, {Michael D.} and Narhi, {Linda O.} and Chang, {Wen Chang} and Lu, {Hsieng S.}",
year = "1996",
month = "6",
day = "4",
doi = "10.1074/jbc.271.19.11301",
language = "English",
volume = "271",
pages = "11301--11308",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "19",

}

TY - JOUR

T1 - Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli

AU - Jones, Michael D.

AU - Narhi, Linda O.

AU - Chang, Wen Chang

AU - Lu, Hsieng S.

PY - 1996/6/4

Y1 - 1996/6/4

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=15844416254&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=15844416254&partnerID=8YFLogxK

U2 - 10.1074/jbc.271.19.11301

DO - 10.1074/jbc.271.19.11301

M3 - Article

VL - 271

SP - 11301

EP - 11308

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 19

ER -