重组人磷脂爬行酶1的原核表达及纯化

Prokaryotic Expression and Purification of Recombinant Human Phospholipid Scramblase 1

目的:建立重组人磷脂爬行酶1(hPLSCR1)原核表达及纯化工艺。方法:PCR扩增hPLSCR1编码基因并连接到原核表达载体pET-28a,转化大肠杆菌BL21(DE3)并进行诱导表达,Western印迹鉴定所表达蛋白;优化表达条件后,Ni2+柱亲和层析纯化重组蛋白。结果:构建了pET-28a-PLSCR1重组质粒,诱导表达后,经SDS-PAGE分析目的蛋白的表达量达32%,Ni2+金属螯合法纯化目的蛋白后纯度达到95%以上,Western印迹验证了融合蛋白的特异性。结论:建立了高效稳定的His-PLSCR1表达体系,获得大规模生产His-PLSCR1的分离纯化工艺,为进一步研究其蛋白功能奠定了基础。

Objective： To express and purify the recombinant human phospholipid scramblase 1（hPLSCR1）.Methods： The coding sequence of hPLSCR1 was amplified by PCR,and it was cloned into the vector of pET-28a to construct the recombinant plasmid pET-28a-PLSCR1.The recombinant E.coli BL21（DE3）/pET-28a-PLSCR1 was induced by IPTG,the recombinant protein was identified by Western blot.Optimized the terms of expressiom,the fusion protein was purified by nickel-chelating chromatography.Results： Recombinant plasmid pET-28a-PLSCR1 was constructed,and purified fusion protein His-PLSCR1 was successfully obtained.Then induction were optimized.Over 32% expression level was achieved as analyzed by SDS-PAGE.After being purified,the purity of expressed product reached above 95%.Western blot analysis showed that the fusion protein was specificity.Conclusion： The expression system of hPLSCR1 was established,which can be applied to further studies of the function of this protein and further research on pathogenesis.