Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.
Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.
Watanabe
T
T
Biotechnology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Seno
M
M
Sasada
R
R
Igarashi
K
K
D002455:Cell Division / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000378:metabolism; D004727:Endothelium / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism*; D016220:Fibroblast Growth Factor 1 / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006493:Heparin / Q000378:metabolism; D006801:Humans; D009389:Neovascularization, Pathologic; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013053:Spectrophotometry
D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D003300:Copper; D006493:Heparin