Through site-directed mutagenesis, three cysteines of human basic fibroblast growth factor (hbFGF) were replaced with serine residues, resulting in a hbFGF mutant named hbFGF(Ser25,69,92). 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 (PEG(5K)) via the cysteine residue to obtain another hbFGF derivative, PEG(5K)-hbFGF(Ser25.69.92). The optimal modification reaction was conducted at 4 degrees C for 4 h at a molar ratio of PEG(5K) to hbFGF(Ser25,69,92) 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 PEG(5K)-hbFGF(Ser25,69,92) 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. (c) 2006 Elsevier Inc. All rights reserved.