Research On The Stability Of Recombinant Human Basic Fibroblast Growth Factor In Vitro

Object: To investigate the mechanism of aggregating of rhbFGF protein in solution and the possibility of the aggregating degree decreasing by site-directed mutagenesis,we analysis the affecting factor of aggregating of rhbFGF protein in solution and compare the difference of rhbFGF(w) and rhbFGF(m) in aggregating;While screening some universial addictive to improve the solubility of hbFGF protein in solution and applying them to practice,so as to further better the stability of rhbFGF in the producing process.Method: By the solubility of hbFGF protein in solution and its mitogen in vitro as marker to indicate the difference of bFGF(w) and bFGF(m) ,which is in different salts and different pH system ,while comparing the difference on stability of different concentration protein and different concentration salts in solution; besides,we use non-reduced SDS-PAGE and HPLC method to analysis the component of supernant of bFGF solution and calculating the surface tension of the solution of adding different substance to explore the mechanism of the stability of rhbFGF protein in solution.Results: hbFGF(m) is 1. .Under the same conditions(pH7.0,NaC12.0M),we can find that the aggregating degree of bFGF(w) is obviously higher than bFGF(m) at first stage and there are all some soluble aggregation in their supernant; under the same conditions,the solubility and biological activity of hbFGF(m) which is added two type sugars and two type polyols is increased with their concentration ,the largest solubility and biological activity of hbFGF(m) observed in 0.8M of sucrose and trehalose and in 6%(v/v) of glycerol and ethylene glycol,the protein concentration of hbFGF(m) is 1.145±0.0072mg/ml (sucrose), 1.125±0.0056mg/ml (trehalose), 0.831±0.0082 ( glycerol ) , 1.053±0.0097 ( ethylene glycol ) , and biological activity is ED₅₀=0.48ng/ml and ED₅₀=0.58ng/ml and ED50=2.25ng/ml and ED50=1.85ng/mlrespectively.Conclusion:.Under the same conditions,the degree of aggregating and the speed of aggregation of hbFGF(w) is higher than hbFGF(m). we find that aggregation depend on protein concentration,the higher bFGF in solution,the more polymer.Disulfide and hydrophobic interaction play marjor roles in the aggregating process of hbFGF in solution and it can decrease the aggregation of hbFGFwhen Cys sites is replaced by serines by site-directed mutagenesis.Trehalose and sucrose can prevent the aggregation of hbFGF(m) and increase its solubility in solution and the biological activity of hbFGF(m) in unit volume solution,so do glycerol and ethylene glycol. This will facilitate our further research in meliorating the stability of hbFGF in solution.