Properties of Variant Forms of Human Stem Cell Factor Recombinantly Expressed in Escherichia coli

Langley, Keith; Mendiaz, Elizabeth A.; Liu, N.L.; Narhi, Linda O.; Zeni, Luigi; Parseghian, C.M.; Clogston, Christi L.; Leslie, I.; Pope, Joseph A.; Lu, Hsieng S.; Zsebo, KM; Boone, Thomas C.

doi:10.1006/abbi.1994.1208

Cited by 49 publications

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“…Since SCF 1-141 is fully active (29) and is a non-covalently associated dimer as judged by size exclusion chromatography, the Ala 165 almost certainly does not reside in the dimer interface. The C-terminal portion, which would include the disulfide bond Cys 165 -Cys 165 of the SCF(A165C) disulfide-linked dimer, is probably flexible.…”

Section: Discussionmentioning

confidence: 99%

The Majority of Stem Cell Factor Exists as Monomer under Physiological Conditions

Hsu

Mendiaz

et al. 1997

Journal of Biological Chemistry

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Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (K a ) of 2-4 ؋ 10 8 M ؊1 , using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization K a , greater than 90% of the circulating SCF would be in the monomeric form. When 125 I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10 -100 ng/ml, consistent with the K a determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer K a values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH 2 CONH 2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys 165 -Cys 165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.Stem cell factor is a cytokine that is active toward early hematopoietic cells and also plays roles in gametogenesis, melanogenesis, and mast cell function. Its biological and other properties have been extensively reviewed (1, 2). It is found in both membrane-bound and soluble forms, with the latter being derived from a membrane-bound form by proteolytic cleavage. The soluble SCF has 165 amino acids.Both soluble Escherichia coli-derived and CHO 1 cell-derived recombinant human SCF have been reported to be non-covalently associated dimers, as determined by sedimentation equilibrium and size exclusion chromatography at protein concentrations above 0.4 mg/ml (3). In a previous paper (4), we demonstrated that SCF dimer is dissociable under non-denaturing conditions and the dissociation rate constant (k d ) of E. coli-derived rhSCF dimer is approximately 1.35 ϫ 10 Ϫ4 s Ϫ1 at pH 4.8, 25°C. In the present work, we arrive at a value of 2-4 ϫ 10 8 M Ϫ1 for the dimer association constant (K a ) of E. coli-derived rhSCF, based on several approaches including ultracentrifugation and size exclusion chromatography at low SCF concentrations. Since the SCF concentration in human serum has been found previously to be a few nanograms/ml (5), the K a value suggests that the majority of SCF in serum may be monomeric. We use 125 I-SCF as a tracer added to serum to show that this does in fact appear to be the case.The binding of ligands to cell receptors, followed by receptor dimerization, is essential for signal transduction by the family of transmembrane receptor tyrosine kinases (6 -8). The receptor for SCF on target cells is Kit (see Refs....

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Section: Discussionmentioning

confidence: 99%

The Majority of Stem Cell Factor Exists as Monomer under Physiological Conditions

Hsu

Mendiaz

et al. 1997

Journal of Biological Chemistry

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“…The Kitl sequences represent the functional core of Kitl that contains the dimer interface and the domain involved in Kit receptor binding and are sufficient to mediate cell proliferation activity (Langley et al, 1994). In addition, previous reports have identified a major cleavage site at A164 or A165 (Zsebo et al, 1990), and two additional minor secondary cleavage sites near S142 and KAAK177-180 (Cheng and Flanagan, 1994;Majumdar et al, 1994).…”

Section: Evaluation Of the Effect Of Deletions On Kitl Sheddingmentioning

confidence: 99%

Different ADAMs have distinct influences on Kit ligand processing: phorbol-ester-stimulated ectodomain shedding of Kitl1 by ADAM17 is reduced by ADAM19

Kawaguchi

Horiuchi

Becherer

et al. 2007

Journal of Cell Science

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Kit ligand (Kitl), the ligand for the Kit receptor tyrosine kinase, plays important roles in hematopoiesis, gametogenesis and melanogenesis. Kitl is synthesized as a membrane-anchored precursor that can be processed to produce the soluble growth factor. Here, we evaluated the role of ADAM (a disintegrin and metalloprotease) metalloproteases in ectodomain shedding of Kitl. We found that both ADAM17 and ADAM19 affect Kitl1 shedding, albeit in different ways. Overexpression of ADAM19 resulted in decreased levels of Endo-H-resistant mature Kitl1, thereby reducing the amount of Kitl that is shed from cells following stimulation with phorbol esters. ADAM17 was identified as the major phorbol-ester-stimulated sheddase of Kitl1, whereas ADAMs 8, 9, 10, 12 and 15 were not required for this process. ADAM17 also emerged as the major constitutive and phorbol-ester-stimulated sheddase of Kitl2 in mouse embryonic fibroblasts. Mutagenesis of the juxtamembrane domain of Kitl2 showed no stringent sequence requirement for cleavage by ADAM17, although two nonadjacent stretches of four amino acid residues were identified that are required for Kitl2 shedding. Taken together, this study identifies a novel sheddase, ADAM17, for Kitl1 and Kitl2, and demonstrates that ADAM19 can reduce ADAM17-dependent phorbol-ester-stimulated Kitl1 ectodomain shedding.

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“…To date, SCF has exclusively been expressed in E. coli as insoluble and inactive aggregates (Arakawa et al, 1991;Langley et al, 1994;Langley et al, 1992;Lili et al, 2006;Martin et al, 1990;Wang et al, 2008;Wang et al, 2004). The current strategy for recovery of active SCF from inclusion bodies includes solubilization, oxidation and refolding steps (Wang et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner

Bals

Schambach

Meyer

et al. 2011

Journal of Biotechnology

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Properties of Variant Forms of Human Stem Cell Factor Recombinantly Expressed in Escherichia coli

Cited by 49 publications

References 0 publications

The Majority of Stem Cell Factor Exists as Monomer under Physiological Conditions

The Majority of Stem Cell Factor Exists as Monomer under Physiological Conditions

Different ADAMs have distinct influences on Kit ligand processing: phorbol-ester-stimulated ectodomain shedding of Kitl1 by ADAM17 is reduced by ADAM19

Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner

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