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目的构建钙调蛋白3种镁离子结合位点突变体的质粒载体,并进行表达纯化及鉴定,为深入研究钙调蛋白的生物学功能奠定基础。方法利用钙调蛋白cDNA进行点突变,制备3种镁离子结合位点突变的cDNA。将突变后的cDNA分别插入pGEX-6P-3载体,制备钙调蛋白突变体载体质粒。质粒转化大肠杆菌BL21感受态细胞,培养大肠杆菌,并诱导GST融合蛋白表达。利用Glutathione-Sepharose 4B珠子和PreScission蛋白酶分离纯化蛋白。结果酶切鉴定和DNA测序证实成功构建钙调蛋白突变体质粒;表达纯化得到的钙调蛋白突变体经电泳图鉴定纯度较高,浓度约1.0mg/mL。结论本研究成功构建了钙调蛋白镁结合位点突变体融合蛋白原核表达质粒,分离纯化可获得较高浓度较高纯度的钙调蛋白突变体。
OBJECTIVE: To construct a plasmid vector containing three calmodulin binding sites for Mg (superscript 2 +) binding sites, and to express, purify and identify the calmodulin proteins for further study on the biological functions of calmodulin. Methods Mutant cDNA of calmodulin was used to make point mutation. The mutated cDNAs were respectively inserted into the pGEX-6P-3 vector to prepare calmodulin mutant vector plasmids. The plasmid was transformed into E. coli BL21 competent cells, cultured in E. coli, and induced the expression of the GST fusion protein. Purify the protein using Glutathione-Sepharose 4B beads and PreScission protease. Results Restriction endonuclease digestion and DNA sequencing confirmed the successful construction of calmodulin mutant plasmids. The expressed and purified calmodulin mutants were identified by electrophoresis to be highly pure at a concentration of about 1.0 mg / mL. Conclusion The prokaryotic expression plasmid of calmodulin-Mg binding site fusion protein was successfully constructed in this study, and the calmodulin mutants with higher purity and higher purity were isolated and purified.