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KMID : 0385220000100010026
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Korean Journal of Gerontology
2000 Volume.10 No. 1 p.26 ~ p.34
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Expression and Characterization of Soluble Basic Fibroblast Growth Factor and Extracellular Domain of Fibroblast Growth Factor Receptor-1
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Shin Eun-Young
Kim Seok-Yong
Kim Eung-Gook
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Abstract
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Fibroblast growth factor (FGF) and its receptors are involved in tumorigenesis, angiogenesis, and embryonic development. To investigate the effect of this ligand-receptor interaction it is essential to obtain them in a biologically active form. We established an efficient expression system to produce basic FGF (bFGF) in E. coli and extracellular domain of FGF receptor-1 (EC-FGFR) in NIH 373 cells as fusion proteins of thioredoxin-bFGf (TRX-bFGF) and EC-FGFR-alkaline phosphatase (EC-FGFR-AP), respectively. The fusion genes encoding bFGF and EC-FGFR were cloned into the expression vectors, pTRX and pAPtag-l, respectively. The soluble TRX-bFGF was purified to homogeneity with heparin-affinity chromatography. Purified TRX-bFGF induced tyrosine phosphorylation of cellular proteins comparable to the commercially available recombinant bFGF, indicating that the fusion protein exerts its effects. On the other hand, EC-FGFR-AP was partially purified and its identity was confirmed by Western blot. With addition of the culture medium containing EC-FGFR-AP, alkaline phosphatase activity in TRX-bFGF bound heparin-agarose bead increased in a dose-dependent manner, indicating that EC-FGFR-AP and TRX-bFGF can make a complex and its presence is detectible by measurement of the enzyme activity. Our results imply that TRX-bFGF and EC-FGFR-AP can be used as a potential agonist to promote angiogenesis and as a bait to screen the inhibitor of bFGF, respectively.
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KEYWORD
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Basic fibroblast growth factor, Fibroblast growth factor receptor, Expression, Purification, Characterization
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