LSECtin-CRD-GST融合蛋白的原核表达、纯化及复性  

Prokaryotic Expression,Purification and Refolding of LSECtinCRD-GST

作　　者：汪明群[1] 丛建波[2] 程莉[2] 董国福[2] 王少霞[2] 郑文[2] 王长振[2] 吴可[2] 

机构地区：[1]宜宾市第一人民医院,四川宜宾644000 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《生物技术通讯》2015年第5期658-660,共3页Letters in Biotechnology

基　　金：国家自然科学基金(31170714);北京市自然科学基金(7132134)

摘　　要：目的:构建C型凝集素LSECtin主要功能结构域CRD的原核表达载体,在大肠杆菌中表达LSECtin-CRD-GST融合蛋白。方法:根据Gen Bank发布的LSECtin基因序列设计引物,利用基因重组技术将获得的LSECtin-CRDc DNA定向克隆至C端带GST蛋白标签序列的融合表达载体p GEX-6p-1中,转化大肠杆菌Origami(DE3)进行重组蛋白的诱导表达,用GST柱亲和纯化融合蛋白。结果:获得了原核表达载体p GEX-6p-1-LSECtin-CRD,诱导表达出大量相对分子质量约40×103的包涵体融合蛋白,经纯化、复性获得可溶蛋白,经Western印迹鉴定为目的蛋白。结论:获得足量的LSECtin-CRD-GST融合蛋白,为进一步研究CRD蛋白结构域的动态构象变化提供了实验材料。Objective： To construct the prokaryotic expression vector of pGEX-6p-1-LSECtin-CRD and obtain LSECtin-CRD-GST fusion protein from Escherichia coli, then try to get enough soluable expression products. Meth- ods： According to the sequence of LSECtin published in GenBank, the primers were designed, and gene of LSEC- tin was amplified by PCR and cloned to fusion expression-vector pGEX-6p-1. The fusion expression-vector pGEX- 6p-I-LSECtin-CRD was transformed into E.coli Origami（DE3）. The expression product containing GST tag was pu- rified by glutathione affinity chromatography resin after refolding. Results： The fusion expression vector pGEX-6p- 1-LSECtin-CRD was constructed. The fusion protein with molecular of 40 kD was expressed efficiently in inclu- sion bodies in host cells. The final fusion protein was confirmed by Western blotting. Conclusion： The consider- able amount of soluble LSCEtin-CRD-GST fusion proteins were collected, which laid a foundation for the further study of the mobility and conformation changes of LSECtin-CRD.

关 键 词：LSECtin-CRD-GST融合蛋白 原核表达 复性 

分 类 号：Q78[生物学—分子生物学]