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.
Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, PR China.
Xiaoping
X
Wu
Xiaokun
X
Li
Yaoying
Y
Zeng
Qing
Q
Zheng
Sixian
S
Wu
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