Design of recombinant stem cell factor-macrophage colony stimulating factor fusion proteins and their biological activity in vitro.
Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of approximately 84 KD under non-reducing conditions and a monomer of approximately 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.
Chen
Tao
T
School of life science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093, Nanjing, People's Republic of China.
Yang
Jie
J
Wang
Yuelang
Y
Zhan
Chenyang
C
Zang
Yuhui
Y
Qin
Junchuan
J
D000818:Animals; D002460:Cell Line; D003198:Computer Simulation; D017076:Computer-Aided Design; D019281:Dimerization; D015195:Drug Design; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D018411:Spodoptera; D019089:Stem Cell Factor / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D014162:Transfection
D008024:Ligands; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D016173:Macrophage Colony-Stimulating Factor