Expression and characterization of fibroblast growth factor 8 from Mexican axolotl, Ambystoma mexicanum.
Fibroblast growth factor (FGF) has been known to regulate the proliferation and differentiation of a variety of cell types via interaction with a specific FGF receptor on the cell surface. In the present study, Fgf8 cDNA of Mexican axolotl, Ambystoma mexicanum, was expressed in Escherichia coli as an MBP-FGF8 fusion protein. The cell proliferation activity of the recombinant FGF8 (rFGF8) was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assay. The addition of rFGF8 to the culture medium enhanced proliferation of BALB/c 3T3 and BHK21 cells about 1.4-1.5 fold. To analyze the binding activity of rFGF8 to the cell surface, cell surface enzyme linked immunosorbent assay was developed. Comparison of the structure of basic FGF with the computer-simulated structure of FGF8 suggested that Tyr-58, Glu-132, Tyr-139, and Leu-179 might be the potential receptor binding sites. Amino acid substitution muteins of FGF8 were constructed by PCR-derived directed mutagenesis and the muteins were overexpressed in E. coli. The rFGF8 muteins were purified and their binding activities were analyzed. Substitution of Tyr-58 or Glu-132 or Leu-179 of the FGF8 with alanine reduced the binding affinity, while substitution of Tyr-139 with alanine did not alter the binding affinity. These results imply that Tyr-58, Glu-132, and Leu-179 of FGF8 might be involved in its binding to the cell surface.
Lee
S Y
SY
Department of Life Science, Sogang University, Seoul, Korea.
Kim
W S
WS
Yang
J M
JM
D016475:3T3 Cells; D000558:Ambystoma mexicanum / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism
D011956:Receptors, Cell Surface; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors