Abstract
The extracellular domain of c-kit, which is the receptor for stem cell factor (SCF), was fused genetically to the Fc portion of human immunoglobulin G1. This chimeric protein, c-kitFc, was then expressed in the baculovirus system. The fusion product was secreted into the serum-free culture medium as a soluble dimeric form of approximately 210 Kda. The recombinant protein was easily purified by protein A affinity chromatography at approximately 25 micrograms protein per ml of medium. Binding assay and cross-linking assay showed that the fusion protein retained high affinity for binding SCF (Kd = 0.3 nM). Addition of the chimeric protein into the culture medium of SCF-dependent cells inhibited cell proliferation in a dose-dependent manner. These results suggest that the dimeric c-kitFc protein can be used as an antagonist of SCF for the study of hematopoietic progenitor cells.
MeSH terms
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Animals
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Baculoviridae
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Cell Line
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Chromatography, Affinity
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Culture Media, Serum-Free
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Hematopoietic Cell Growth Factors / metabolism
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Humans
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Immunoglobulin Fc Fragments / biosynthesis*
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Immunoglobulin Fc Fragments / isolation & purification
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Immunoglobulin G / biosynthesis*
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Immunoglobulin G / isolation & purification
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Kinetics
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Molecular Weight
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Moths
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Proto-Oncogene Proteins / biosynthesis*
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Proto-Oncogene Proteins / isolation & purification
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Proto-Oncogene Proteins c-kit
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Receptor Protein-Tyrosine Kinases / biosynthesis*
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Receptor Protein-Tyrosine Kinases / isolation & purification
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Receptors, Colony-Stimulating Factor / biosynthesis*
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Receptors, Colony-Stimulating Factor / isolation & purification
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Recombinant Fusion Proteins / biosynthesis*
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Stem Cell Factor
Substances
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Culture Media, Serum-Free
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Hematopoietic Cell Growth Factors
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Immunoglobulin Fc Fragments
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Immunoglobulin G
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Proto-Oncogene Proteins
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Receptors, Colony-Stimulating Factor
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Recombinant Fusion Proteins
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Stem Cell Factor
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Proto-Oncogene Proteins c-kit
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Receptor Protein-Tyrosine Kinases