Establishment Of A Hypoglycemic Agents Screening Method Based On Human Pancreatic Glucokinase

Human pancreatic glucokinase(PGK),a member of the hexokinase family,is found in the liver,where it participates in the control of blood glucose homeostasis, and in the pancreatic beta cells,where it serves as glucose sensor and regulates insulin release.These particular features make PGK a strong potential target for the pharmacological treatment of type 2 diabetes.Recently,the research of screening glucokinase activators(GKAs) both at home and abroad has become popular and the topic for treating and prevertion of diabetes mellitus.Therefore,it is necessary to construct a reliable and sensitive high-throughput way for screening GKAs in vitro.In this paper,human PGK was successfully expressed in a prokaryotic expression system,and purified as histidine-tagged fusion protein.Eventually,we established a reliable platform for screening GKAs in vitro based on PGK.The main contents are illustrated as following:1.The gene coding human pancreatic glucokinase protein was amplified by polymerase chain reaction(PCR),then was inserted into an expression vector 6HisT-pRSET to get recombinant plasmid.The recombinant plasmid 6HisT-pRSET-PGK was transformed into E.coli BL21 and induced by IPTG..The molecular mass of expressed product,which is in the supernatant,is 55 KDa indentified by SDS-PAGE.2.Recombinant PGK with 6His·Tag was purified by Ni-NTA His·Bind Resin. The purity of PGK is nearly 100%analyzed by SDS-PAGE and Quantity One software.The concentration of recombinant protein is 2.07 mg/mL determined by Bradford method.3.Coupled assay systems was used to assay the enzymatic activity of recombinant PGK.According to the increase in OD_340nm for approximately 10 min at 30℃,pH 9.0 of both the test and black,the specific activity of the purified PGK was 3.77 U/mg.4.Response surface methodology(RSM) was used to optimize the microplate-based GKAs screening platform.The procedure of reaction is:As little as 150μL assay system with the combination of 60 mM Tris,2.11 mM MgCl₂,4.0 mM ATP,6.0 mMβ-D(+)Glucose,1.0 mMβ-NADP,20 U/L G-6-PDH and 20 U/L PGK was incubated at 37℃,pH 7.0 for 13.7 min and measured at 340 nm.Under the optimal conditions,the predicted value of maximum activity ratio and the practical activity ratio was 34.1%and 34.8%respectively.This result corroborated the validity and the effectiveness of this model.the determination coefficient(R²) of multiple regression analysis of the experimental data by Design-Expert 6.0.5 was 0.9442,which ensures adequate credibility of the model.