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Objective To establish a reliable platform for screening glucokinase activators(GKAs)in vitro.Methods Pancreatic glucokinase(PGK)protein expressed in a prokaryotic expression system as a histidine-tagged fusion protein from Homo sapiens was produced.Then,response surface methodology(RSM)was used to optimize the microplate-based GKA screening platform.In the first step of optimization with Plackett-Burman design(PBD).initial pH,reaction time and MgCl_2 were found to be important factors affecting the activity ratio of GKA(RO-28-1675)significantly.In the second step.a 2~3 full factorial central composite design(CCD) and RSM were applied to the optimal condition determination of each significant variable.A second-order polynomial was determined by a multiple regression analysis of the experimental data.Results The following optimal values for the critical factors were obtained:initial pH 0(7.0),reaction time-0.63(13.7 min)and MgCl_2 0.11(2.11 mmol/L) with a predicted value of the maximum activity ratio of 34.1%.Conclusion Under the optimal conditions,the practical activity ratio is 34.8%.The determination coefficient(R~2)is 0.9442,ensuring adequate credibility of the model. LLAE3.extracted from Folium nelumbinis in our laboratory,has prominently activated effects on PGK.
Objective To establish a reliable platform for screening glucokinase activators (GKAs) in vitro. Methods Pancreatic glucokinase (PGK) protein expressed in a prokaryotic expression system as a histidine-tagged fusion protein from Homo sapiens was produced. Chen, response surface methodology (RSM) was used to optimize the microplate-based GKA screening platform. In the first step of optimization with Plackett-Burman design (PBD). inial pH, reaction time and MgCl_2 were found to be important factors affecting the activity ratio of GKA (RO-28 -1675). In the second step. A 2 ~ 3 full factorial central composite design (CCD) and RSM were applied to the optimal condition determination of each significant variable. A second-order polynomial was determined by a multiple regression analysis of the experimental data. Results The following optimal values for the critical factors were obtained: initial pH 0 (7.0), reaction time-0.63 (13.7 min) and MgCl_2 0.11 (2.11 mmol / L) with a predicted value of the maximum Activity ratio of 34.1% .Conclusion Under the optimal conditions, the practical activity ratio is 34.8%. The determination coefficient (R ~ 2) is 0.9442, which is an adequate credit of the model. LLAE3.extracted from Folium nelumbinis in our laboratory, has prominently activated effects on PGK.