Abstract
Distinct from the noncovalently linked recombinant human stem call factor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF. Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds. The cysteines are paired as in the noncovalently associated dimer except that all pairings are intermolecular rather than intramolecular. Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out. The molecule behaves similarly to the noncovalently associated dimer during ion-exchange or gel permeation chromatography. However, the disulfide-linked dimer exhibits increased hydrophobicity in reverse-phase columns and in the native state does not undergo spontaneous dimer dissociation-association as seen for the noncovalent dimer. Spectroscopic analyses indicate that the disulfide-linked and noncovalently associated rhSCF dimers have grossly similar secondary and tertiary structures. In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched human CD34+ cells. The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer. Formation of intermolecular disulfides in the disulfide-linked dimer with retention of biological activity has implications for the three-dimensional structure of noncovalently held dimer and disulfide-linked dimer.
Highlights
Distinct from the noncovalently linked recombinant human stem cell factor dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF
Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds
Active rhSCF can be recovered after folding and oxidation of inactive rhSCF in the solubilized extracts prepared from inclusion bodies after recombinant expression in bacteria [17, 18, 23]
Summary
Materials—E. coli-derived rhSCF (SDS-dissociable dimer) was purified according to methods described previously [17, 18]. Isolation of SDS-nondissociable Dimer—Recovery of rhSCF expressed in E. coli includes solubilization of rhSCF-containing inclusion bodies, oxidation, and folding and subsequent chromatographic steps. N-terminal amino acid sequence analysis of peptides was performed on an automatic protein sequencer (Applied Biosystems models 477A and 470 or Hewlett Packard HPG1000A) as described elsewhere [12]. Chemical Cleavages and Analysis of Cleavage Products—Cleavage of rhSCF dimer species by BNPS-skatole (Pierce) was carried out in 50% acetic acid as described elsewhere [12]. This reaction allows the cleavage of the sample at Trp, which is the lone Trp in each monomer. BFU-E assays were performed using procedures identical to GM-CFC assays except that EPO (4 units/ml) was added to the cultures instead of rhG-CSF
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