Establishment of Hypoglycemic Agent Screening Method Based on Human Glucokinase  

作　　者：CHOU-FEI WU,YANG XU~2,YONG TAO,AND JI-YAN YANG Key Laboratory of State Food Science and Technology,Jiangxi-OAI Joint Research Institute, Nanchang University,Nanchang 330047,Jiangxi,China 

出　　处：《Biomedical and Environmental Sciences》2009年第1期62-69,共8页生物医学与环境科学（英文版）

基　　金：supported by the Program for Changjiang Scholars and Innovative Research Team in University,PCSERT(No.IRT0540).

摘　　要：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 f＇trst step of optimization with Plackett-Burman design （PBD）, initial pH, reaction time and MgC12 were found to be important factors affecting the activity ratio of GKA （RO-28-1675） significantly. In the second step, a 23 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 MgC12 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 （R2） 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. Then, response surface methodology （RSM） was used to optimize the microplate-based GKA screening platform. In the f＇trst step of optimization with Plackett-Burman design （PBD）, initial pH, reaction time and MgC12 were found to be important factors affecting the activity ratio of GKA （RO-28-1675） significantly. In the second step, a 23 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 MgC12 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 （R2） is 0.9442, ensuring adequate credibility of the model. LLAE3, extracted from Folium nelumbinis in our laboratory, has prominently activated effects on PGK.

关 键 词：Screening mothod Human pancreatic glucokinase Protein expression Glucokinase activators Response surfacemethodology 

分 类 号：R-331[医药卫生] R96