Purification and modification by polyethylene glycol of a new human basic fibroblast growth factor mutant-hbFGFSer25,87,92

The mutant of human basic fibroblast growth factor (hbFGF), hbFGFSer25,87,92, which was constructed by replacing the cysteine residues at the positions of the 25th, the 87th and the 92nd with serine residues, was coupled to polyethylene glycol (PEG) with a molecular size of 20 kDa (20 K) (PEG20K) to obtain hbFGF derivative, PEG20K-hbFGFSer25,87,92. The optimal modified reaction was conducted at 12 °C for 12 h with the molar ratio of PEG20K to hbFGFSer25,87,92 of 30:1. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the modification rate was up to 60%. The PEGylated product retained binding affinity to heparin and could be purified by heparin affinity chromatography. Compared to hbFGF mutant, purified PEG20K-hbFGFSer25,87,92 retained about 34% of mitogenic activity. Heat-stability assay indicated that the modified product was more stable than the native protein at the temperature of 37 °C.