Wu Xiaoping, Li Xiaokun, Zeng Yaoying, Zheng Qing, Wu Sixian
Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, PR China.
Protein Expr Purif. 2006 Jul;48(1):24-7. doi: 10.1016/j.pep.2006.02.002. Epub 2006 Feb 28.
Through site-directed mutagenesis, three cysteines of human basic fibroblast growth factor (hbFGF) were replaced with serine residues, resulting in a hbFGF mutant named hbFGFSer25,69,92. The mutant with only one cysteine residue at the 87th position, whose mitogenic activity was comparable to that of wild-type hbFGF, was further coupled to polyethylene glycol with a molecular size of 5 kDa (PEG5K) via the cysteine residue to obtain another hbFGF derivative, PEG5K-hbFGFSer25,69,92. The optimal modification reaction was conducted at 4 degrees C for 4 h at a molar ratio of PEG5K to hbFGFSer25,69,92 of 20:1. The result of SDS-PAGE showed that the modification extent was up to 80%. The modified product was purified by ion exchange chromatography. Compared to the hbFGF mutant, the purified PEG5K-hbFGFSer25,69,92 still retained about 60% of the mitogenic activity of the former, which provided a good basis for further studying the bioactivity of the PEGylated protein in vivo.
通过定点诱变，将人碱性成纤维细胞生长因子（hbFGF）的三个半胱氨酸替换为丝氨酸残基，得到一种名为hbFGFSer25,69,92的hbFGF突变体。该突变体在第87位仅含有一个半胱氨酸残基，其促有丝分裂活性与野生型hbFGF相当，通过该半胱氨酸残基进一步与分子量为5 kDa的聚乙二醇（PEG5K）偶联，得到另一种hbFGF衍生物PEG5K-hbFGFSer25,69,92。最佳修饰反应在4℃下以PEG5K与hbFGFSer25,69,92的摩尔比为20:1进行4小时。SDS-PAGE结果表明修饰程度高达80%。修饰产物通过离子交换色谱法进行纯化。与hbFGF突变体相比，纯化后的PEG5K-hbFGFSer25,69,92仍保留了前者约60%的促有丝分裂活性，这为进一步研究聚乙二醇化蛋白在体内的生物活性提供了良好基础。
Zotero 插件