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The amino acid at the 119th position of human basic fibroblast growth factor(hbFGF),lysine(K119),is a critical component for its mitogenic activity.However,little is known about the effects of the characteristics of this residue including charge on the mitogenic activity of hbFGF.Herein,this basic residue was replaced with neutral glutamine residue and acidic glutamic acid residue to construct mutants hbFGF~(K119Q) and hbFGF~(K119E),respectively.The mutants were produced by BL21(DE3)/pET3c expression system and purified to homogeneity by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. The mitogenic activity analysis showed that neutralization of charge at the 119th residue diminished the mitogenic activity of hbFGF,whereas change of positive charge to negative charge at this residue had no significant effect on its mitogenic activity. Further MAP kinase activation assay revealed that the influence of different charge at the 119th position on the mitogenic activity of hbFGF was related to the signal molecular activation in MAPK pathway.It was deduced that the charge,either positive or negative, at the 119th position of hbFGF is crucial for its full mitogenic activity.
The amino acid at the 119th position of human basic fibroblast growth factor (hbFGF), lysine (K119), is a critical component for its mitogenic activity. However, little is known about the effects of the characteristics of this residue including charge on the mitogenic activity of hbFGF.Herein, this basic residue was replaced with neutral glutamine residue and acidic glutamic acid residue to construct mutants hbFGF ~ (K119Q) and hbFGF ~ (K119E), respectively.The mutants were produced by BL21 (DE3) / pET3c expression system and purified to homogeneity by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. The mitogenic activity analysis showed that neutralization of charge at the 119th residue diminished the mitogenic activity of hbFGF, Where change of positive charge to negative charge at this residue had no significant effect on its mitogenic activity. Further MAP kinase activation assay revealed that the influence of different charge at the 119th position on the mitogenic activity of hbFGF was related to the signal molecular activation in MAPK pathway. It was deduced that the charge, either positive or negative, at the 119th position of hbFGF is crucial for its full mitogenic activity.