Site-directed PEGylation of human basic fibroblast growth factor.
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.
Wu
Xiaoping
X
Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, PR China.
Li
Xiaokun
X
Zeng
Yaoying
Y
Zheng
Qing
Q
Wu
Sixian
S
D000818:Animals; D041702:BALB 3T3 Cells; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D011092:Polyethylene Glycols; D012694:Serine; D003545:Cysteine