大鼠CRIP2蛋白的表达和分离纯化

Expression and Purification of Rat Cysteine-Rich Intestinal Protein 2

目的:获得大鼠crip2基因片段,并在大肠杆菌中表达、纯化大鼠CRIP2(cysteine-rich intestinal protein 2)蛋白。方法:从大鼠主动脉组织中提取总DNA,RT-PCR扩增出相应大小的crip2 DNA片段,与pGEM-T-easy载体连接后测序;将测序正确的crip2按照BamHⅠ和HindⅢ酶切位点克隆入原核表达载体pRSET A,将连接产物转化大肠杆菌BL21,挑出阳性克隆,IPTG诱导表达重组的6×His融合蛋白,通过镍柱进行纯化。结果:PCR获得的crip2序列与GenBank报道的一致(为707 bp);重组融合蛋白在大肠杆菌BL21中以可溶形式高效表达,经SDS-PAGE和Western印迹分析,在相对分子质量为27×103处有特异的蛋白条带,经镍柱纯化后,得到了高纯度的CRIP2融合蛋白。结论:克隆了大鼠crip2基因片段,并在大肠杆菌BL21中高效表达,亲和层析纯化后获得高纯度的CRIP2融合蛋白。

Objective： To obtain crip2（cysteinerich intestinal protein 2） gene segments of rat, express efficient ly in E.coli and purify the target proteins. Methods： The coding region of the rat crip2 cDNA was amplified from rat aorta RNA by RTPCR. After sequenced, crip2 gene segments were inserted into pRSET A and expressed in E.coli BL21. Recombinant 6xHis fused proteins were purified via NiNTA purification system. Results： The length of PCR product was 707 bp and identical with that of GenBank reported. The proteins were expressed in E.coli BL21 as soluble proteins, crip2 gene in pRSET A was predicted to encode a recombinant protein of 208 aa with a molecular weight of about 27 kD. Then the 6xHis fused proteins were purified by affinity chromatography. Con clusion： crip2 gene of rat was successfully amplified and expressed in E.coli BL21. Purified proteins were ob tained via NiNTA purification system. This work laid the foundation for the further study of the CRIP2 protein.