Construction of a Mutant CaM-expressing Plasmid,and Expression,Purification,and Activity Identification of the Recombinant Protein / 中国医科大学学报

Objective To construct a CaME141G fusion protein-expressing plasmid,and to express,purify,and identify the activity of the recombinant protein. Methods The 141st site of the wild type CaM,E (GAG),was mutated to G (GGG),using site-specific mutagenesis technology. Escherichia coli BL-21 was transformed with the mutant plasmid. The GST-CaME141G fusion protein was mass-cultured and induced for expression. Subsequently,the GST-CaME141G fusion protein was purified using GS-4B beads. PreScission protease was applied to remove the GST,the Bradford method used to determine the concentration of purified protein,and SDS-PAGE used to detect its relative molecular weight and purity. The GST pull-down assay was used to study the protein's biological activity. Results The CaME141G protein was successfully purified at a high concentration and purity. The protein could interact with PreIQ protein fragments from the myocardial CaV1. 2 calcium channel C terminal,in a CaME141G concentration-dependent manner. Therefore,CaME141G has the ability to bind with the CaV1. 2 calcium channel. Conclusion This study successfully constructed a CaME141G fusion protein-expressing plasmid and purified the CaME141G protein. This lays a foundation for regulating the function of CaM mutations in the myocardial CaV1. 2 calcium channel,and for the study of its relationship with diseases of the cardiovascular system.

Expression and Purification of an N?terminal Fragment of the Cav1.2 Calcium Channel and Characterization of Its Interaction with Calmodulin / 中国医科大学学报

Objective To investigate a method for the purification of the N?terminal peptide fragment(NT)of the myocardial calcium channel Cav1.2,and characterize its interaction with calmodulin(CaM). Methods EscherichiacoliBL?21 cells were transformed with plasmid pGEX?6p?3/NT harboring the NT?GST fusion gene. The cells harboring pGEX?6p?3/NT were cultured and protein expression was induced with isopropyl?β?D?thiogalactoside(IPTG). Then,the GST?NT fusion protein was purified by using glutathione Sepharose 4B(GS?4B)beads. GST was cleaved off with the PreScission protease,and SDS?PAGE was performed to detect the purity and relative molecular weight of the purified peptide. Further, GST pull?down assay was performed to characterize the interaction of the NT peptide with CaM. Results SDS?PAGE analysis showed that the NT peptide was successfully purified,with high purity. Results of the GST pull?down assay showed that the NT peptide could interact with CaM. Conclusion This study establishes a method for the purification of the NT peptide and lays the foundation for further research on the interaction partners and biological functions of NT.

Vector Construction,Protein Expression,Purification,and Identification of Calmodulin Mutants N2 and C2 / 中国医科大学学报

Objective To construct expression vectors of calmodulin(CaM)mutants N2 and C2,and to express,purify,and identify the mutant proteins,in order to study the interactions between CaM and calcium channels. Methods The cDNA of N?lobe and C?lobe of CaM were used to prepare the cDNA of N2 and C2. Next,the recombinant cDNAs were cloned into a pGEX?6p?3 plasmid,and the recombinant plasmids were trans?ferred into E.coli BL21 cells. The transfected BL21 cells were stimulated with IPTG. The fusion proteins were extracted by ultrasonication and puri?fied by using GS?4B beads. Finally,protein activity was identified by the pull?down assay. Results Both the restriction digestion map and the DNA sequence identification results confirmed that the recombinant plasmids were successfully constructed. SDS?PAGE results showed high purity and concentration of N2 and C2 proteins. Their activities and binding abilities with the calcium channel fragment were confirmed by the pull?down assay.Conclusion In this study,expression vectors of N2 and C2 are successfully constructed,and physiologically active N2 and C2 CaM mutant proteins are obtained.

Construction of and Protein Preparation from a Recombinant Plasmid Containing the Distal Fragment of the Distal C-terminus of the Cav1.2 Channel / 中国医科大学学报

Objective To construct a recombinant plasmid vector containing the distal fragment of the distal C-terminus (dDCT) of the Cav 1.2 channel,and express,extract,and purify dDCT protein and characterize its biological activity.Methods dDCT cDNA was ligated into the pGEX-6p-1 vector to create a recombinant plasmid that was subsequently transformed into Escherichia coli BL21 competent cells.Expression of GST-dDCT fusion protein from this plasmid was induced with isopropy-β-D-thiogalactoside,and the resulting protein was purified using glutathione-sepharose 4B beads.The biological activity of dDCT was analyzed by GST pull-down assay.Results The recombinant plasmid was verified by restriction enzyme digestion and sequencing.The concentration and purity of the dDCT protein,which was extracted by ultrasonication,were high enough to detect dDCT activity.The binding of dDCT to CT1 was determined to be concentration-dependent.Conclusion The recombinant dDCT plasmid was successfully constructed,providing the fundamental basis for future studies on mechanisms of Cav 1.2 channel autoregulation.

Estrogen exerts the effect on stress-induced senescence of vascular smooth muscl cells in vitro / 解剖学报

Objective To explore the effects of estrogen on stress-induced senescence of vascular smooth muscle cells (VSMCs) and the underlying mechanisms. Methods The VSMCs of passage 2-3 cultured from female SD rats were induced into senescence by exposing to 150μmol/L H2O2 in the presence or absence of different concentrations(10-10mol/L-10-8mol/L) of 17β-estradiol (E2). The expressions or activities of senescence associated marker DcR2, senescence-associated beta-galactosidase (SA-β-Gal), oncogene Ras and p21WAF1 were detected by flow cytometry, cytochemical staining, pull-down assay or Western blotting analysis. Results Flow cytometry analysis showed that in the physiological concentrations, E2 significantly inhibited the H2O2-promoted high-level expression of DcR2 of VSMCs in a dose-dependent manner, with a highest inhibitive rate at 14.48%±0.6%(E2=10-8 mol/L;P<0.05, n =3);this inhibitive effect could be blocked by a E2 receptors inhibitor ICI 182,780. Cytochemistry staining showed that the rate of SA-β-Gal positive VSMCs induced by H2O2 decreased in presence of 10-8mol/L E2 (20.5%±1.4% vs 9.6%±0.9%;P<0.05, n =9). Pull-down assay and Western blotting analysis revealed that administration of 10-8mol/L E2 obviously reduced the H2O2-induced activity of Ras (0.60±0.06 vs 0.26±0.04;P<0.05, n =3) and expression of p21WAF1 (0.46±0.04 vs 0.33±0.02;P<0.05, n =3). Conclusion E2 exerts, an inhibitive effects on stress-induced senescence of VSMCs by suppressing the activity of Ras and expression of p21WAF1. This finding suggests a novel mechanism for the hormone's anti-atheroschlerotic effects.