RESUMEN
Objective To construct plasmid vectors of calmodulin(CaM)Mg2+binding site mutants,and to express,purify and identify the mutant proteins. Methods Three kinds of cDNAs coding for the mutated CaM were cloned into pGEX?6P?3 plasmid vectors. These recombinant plasmids were transfected into Escherichia coli BL21 to express GST fusion proteins of CaM mutants. The fusion proteins were purified with Glutathione?Sep?harose 4B beads and PreScission protease. Results Both enzyme digestion analysis and DNA sequence identification proved the successful con?struction of the CaM mutant plasmids. SDS?PAGE results showed the high purity of each CaM mutant protein. The concentrations of three CaM mu?tants were around 1.0 mg/mL. Conclusion Prokayotic expression vectors of CaM Mg2+binding site mutants were successfully developed,and the eli?gible CaM mutant proteins were obtained. This study provided an important basis for further study on CaM’s biological function.
RESUMEN
Objective: To study the expression and activity of the apoptosis protease-Caspase 3 in(E.coli) BL21(DE3). Methods: The cDNA of Caspase 3 was amplified by PCR and inserted into the plasmid pCMV-Myc,it is then cloned to prokaryotic expression vector pGEX-6p,after which Caspase 3 was induced by IPTG.The protein induced was identified by SDS-PAGE and Western blot. Results: After induced by IPTG for 3 hours,the concentration of Caspase 3 reached the highest level. Conclusion: Active Caspase 3 can be induced within E.coli BL21(DE3),further research can be done about the role of Caspase 3 in apoptosis.