Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells.
A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.
Chen
Tao
T
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China.
Zang
Yuhui
Y
Zhu
Jie
J
Lu
Haiqin
H
Han
Junhai
J
Qin
Junchuan
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D018411:Spodoptera; D019089:Stem Cell Factor / Q000235:genetics*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D016173:Macrophage Colony-Stimulating Factor