Watanabe T, Seno M, Sasada R, Igarashi K
Biotechnology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Mol Endocrinol. 1990 Jun;4(6):869-79. doi: 10.1210/mend-4-6-869.
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.
编码人酸性成纤维细胞生长因子（haFGF）的合成互补DNA（cDNA）在T7启动子的控制下于大肠杆菌中表达。所产生的haFGF使用肝素-琼脂糖柱和苯基-琼脂糖柱进行了广泛纯化。在存在肝素（10 - 50微克/毫升）的情况下，haFGF对3T3细胞和内皮细胞的促有丝分裂活性显著增强，而在未外源添加肝素的鸡胚绒毛尿囊膜上观察到血管生成活性。这种促有丝分裂活性的显著增强在haFGF中特异性观察到，而非人碱性成纤维细胞生长因子（hbFGF）。haFGF的圆二色光谱不受肝素存在的影响。使用肝素亲和高效液相色谱法（HPLC）检测了haFGF对肝素的亲和力，并精确证实其相对低于hbFGF。这些结果表明haFGF被肝素增强，且这种增强不涉及haFGF分子构象的显著变化。使用铜亲和HPLC柱也证实了haFGF对铜的亲和力高于hbFGF。此外，在酸性条件下，haFGF比hbFGF更稳定，并且在肝素存在下进一步稳定。
