PreIQ及其突变体载体构建表达纯化和活性鉴定

Vector Construction,Expression,Purification and Identification of PreIQ and Its Mutant

目的构建心肌L型钙通道片段PreIQ及其突变体与谷胱甘肽转移酶(GST)重组的融合蛋白原核表达载体并进行表达纯化和活性鉴定。方法将PreIQ及其突变体的cDNA插入pGEX-6p-1质粒载体后,转化大肠杆菌BL21感受态细胞,大量培养并利用异丙基硫代-β-D半乳糖苷(IPTG)诱导PreIQ及其突变体的GST融合蛋白表达,GS-4B beads进行分离纯化。采用SDS-PAGE鉴定目的蛋白分子质量和相对纯度。采用Bradford方法测定纯化后蛋白浓度。采用pull-down法检测纯化后蛋白的活性。结果 PreIQ及其突变体融合蛋白得到了大量表达;纯化的PreIQ及其突变体具有较高的纯度;纯化后蛋白能与钙调蛋白(CaM)结合,具有生物活性。结论本研究成功构建了PreIQ及其突变体融合蛋白原核表达载体,获得具有生物活性的PreIQ及其突变体融合蛋白,为深入研究PreIQ的生能学功能奠定基础。

Objective To develop prokaryotic expression vectors for PrelQ and its mutant GST fusion protein expressions, purification and activity identification. Methods cDNAs of PrelQ and its mutant were cloned into pGEX-6p- 1 plasmid vector, and then transformed to the Escherichia coli BL21 component cells. The transformed BL21 cells were stimulated with IPTG for the expression of GST fusion proteins with PreIQ and its mutant. The fusion proteins were purified with Glutathione-Sepharose 4B beads. SDS-PAGE was used to detect the purity and relative molecular weight. Bradford method was used to determine the concentration of pttfified PreIQ and its mutant. The protein activity was examined by pull-down method. Results SDS-PAGE showed high purity of PreIQ and its mutant with the concentration around 2 mg/mL, which was enough for molecular biological and eleetrophysiological studies. The purified PreIQ and its mutant in this study successfully bind to CaM. Conclusion Pmkaryotic expression vec- tors of PreIQ and its mutant fusion proteins were successfully constructed, and bioaetive fusion proteins were obtained. These results provided a basis for further biological function studies of PreIQ.