Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2022198 | Protein Expression and Purification | 2006 | 4 Pages |
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 °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.