Expression, Purification And Characterization Of HsGSK-3β For High-throughput Screening

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase, highly conserved and has been identified in every eukaryote investigated to date. It is encoded by two isoforms in mammals, termed GSK-3 and GSK-3 . GSK-3 plays an important role in at least three signal transductory systems, namely, the Wnt/wingless, Hedgehog and PI' 3 -kinase pathways, which influence proliferation and cell survival, respectively, highlighted by the wide array of substrates controlled by this enzyme that includes cytoplasmic proteins and nuclear transcription factors. Several diease such as in Non-insulin dependent diabetes metillus (NIDDM), Alzheimer's disease, neurological disorders, and cancer have been related to GSK-3, as a potential therapeutic target. Several new GSK-3 inhibitors, such as the aloisines, the paullones and the maleimides, have been developed. Although they are just starting to be characterized in cell culture and animal model experiments, these new inhibitors hold promise as therapeutic agents.Here we reported the cloning, expression, purification and characterization of molecule-based assay for high-throughput inhibitor screening. By screening the compound library of National Center for Drug Screening, we found some hits with novel structure skeleton which should be further studied in cell functions.The coding region of HsGSK-3 was amplified by PCR method from an EST clone containing the HsGSK-3 gene and cloned into the vector pGEX-KG. The recombinant plasmid pGEX-KG/HsGSK-3 was then transformed into E.coli BL21(DE3)/codon plus cells. The GST-HsGSK-3P was overexpressed in the supernatant after IPTG induced. After sonicating, it was first purified by the Glutathione Chromatophy (5 ml) and thrombin cleavage overnight. Then the protein was loaded onto the SP column (5 ml) and eluted by 50 mM - 1 M NaCl gradient. The eluent was hyperfiltered with centrifuge tube and load onto the Q column (1ml). The flow through collected was the purified enzyme.First we ensured the dose-depedent and time-depedent of the enzyme, then optimized the reaction system by Mg2+, DTT dependence, metal chelating agents, ion strength. By these, the opitimized reaction system containes 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mMDTT, SOuMATP, 0.2uCi -33PATP, SOuMCREB, 100 nM /&GSK-3P in 30 ul as the activity assay system. The characterization of the HyGSK-3(3 includes, pH dependence, the kinetics of enzyme reaction and inhibition analysis and enzyme stability. The pH profile data shows that #sGSK-3p is a neutral kinase while the analyses of kinetic constants for synthesized substrates CREB and ATP, inhibition by Aloisnine A indicate that recombinant /M}SK-3p activity retains the enzyme characters of natural /ftGSK-3p\ Enzyme stability analysis shows that the enzyme can be stored at -80 掳C for long time with the solution 50 mM Hepes pH7.2, 100 mM NaCl,. 1 mM DTT and 1 mM EDTA as the storage buffer.According to characterization of HsGSK-3 , the model of high-throughput screening was developed. The 50 ul reaction system included 50 mM Tris-HCl pH7.5, 10 mM MgCl2, 2 mM DTT, 50 uM CREB, 25 uM ATP and 0.15 uCi y-33P ATP, 4% DMSO and 2 ul compound (the final concentration is 40 ug/ml). We established the standard operation procedure of high-throughput screening for GSK-3 inhibitors. 2720 samples of National Center for Drug Screening were screened in 2days and there were 145 samples are higher than 50% inhibition. They were selected to do the second screening with the same compound concentration and 5-fold dilution. While one's inhibitories are both higher than 50%, we calculated 50% inhibitory concentration (IC50) by 3-fold compound dilution up to 0.549ug/ml.From this screening we obtained 6 compounds whose IC50 values are between 10uM and 20uM while 3 compounds whose IC50 values are lower than 10uM. The best inhibitor of HsGSK-3p is named SH00011738, whose IC50 value is 2.24uM and whose molecular mass is 330.36. As the result showed that the screening model is reliable and effective, offering clues for further research on drug discovery and d...