Characterization of a fusion protein composed of the extracellular domain of c-kit and the Fc region of human IgG expressed in a baculovirus system.
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.
Liu
Y C
YC
Pharmaceutical Research Laboratory, Kirin Brewery Co., LTD, Maebashi, Japan.
Kawagishi
M
M
Kameda
R
R
Ohashi
H
H
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002846:Chromatography, Affinity; D016895:Culture Media, Serum-Free; D016298:Hematopoietic Cell Growth Factors / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D009036:Moths; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000302:isolation & purification; D016184:Receptors, Colony-Stimulating Factor / Q000096:biosynthesis* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D019089:Stem Cell Factor
D016895:Culture Media, Serum-Free; D016298:Hematopoietic Cell Growth Factors; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases