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目的构建GRP78原核表达重组质粒pW28-GRP78,在大肠杆菌BL21中表达并纯化获得GRP78蛋白质,检测其ATP酶活性。方法通过NCBI数据库得到GRP78基因序列,PCR扩增获得GRP78基因片段,并插入到带有His标签的载体pW28中,构建重组质粒p W28-GRP78,然后转入大肠杆菌BL21中进行表达,利用镍离子亲和层析纯化目的蛋白。对GRP78进行ATP酶活性检测,并检测GRP78蛋白浓度、反应温度对GRP78蛋白ATP酶活性的影响。结果GRP78蛋白在大肠杆菌BL21中获得大量上清表达,并成功纯化了目的蛋白;ATP酶活性检测实验发现GRP78蛋白具有ATP酶活性,其酶活性与GRP78蛋白浓度、反应温度有关。结论 GRP78蛋白具有ATP酶活性,并且与蛋白浓度、反应温度有关。
Objective To construct the recombinant plasmid pW28-GRP78 of prokaryotic expression plasmid GRP78 and express it in E. coli BL21 to obtain GRP78 protein, and detect its ATPase activity. Methods The GRP78 gene was obtained by NCBI database. The GRP78 gene fragment was amplified by PCR and inserted into the His-tagged vector pW28. The recombinant plasmid pW28-GRP78 was constructed and transformed into E. coli BL21 for expression. Nickel ions Purification of the target protein by affinity chromatography. The activity of GRP78 was detected by enzyme linked immunosorbent assay (ELISA). The effect of GRP78 concentration and reaction temperature on the ATPase activity of GRP78 protein was also tested. Results The GRP78 protein was expressed in E. coli BL21 in a large amount and the recombinant protein was successfully purified. The ATPase activity assay revealed that GRP78 protein possessed ATPase activity. The activity of GRP78 protein was related to the concentration of GRP78 and the reaction temperature. Conclusion GRP78 protein has ATPase activity and is related to protein concentration and reaction temperature.