人Bid蛋白的原核表达及纯化

Prokaryotic Expression and Purification of Human Bid Protein

目的克隆人Bid基因,原核表达并纯化目的蛋白。方法用PCR方法从HeLa细胞cDNA文库中扩增人Bid基因,克隆至pGEX-6P-1表达载体,构建重组表达质粒pGEX-6P-1-Bid,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经GST亲和层析纯化。结果PCR扩增得到588bp的DNA片段,重组表达质粒pGEX-6P-1-Bid经双酶切鉴定和测序表明构建正确。表达的目的蛋白相对分子质量约48000,表达量约占菌体总蛋白的50%,纯化后纯度可达97%。结论已成功克隆并原核表达了人Bid基因,得到纯度较高的Bid蛋白,为进一步研究其结构和功能奠定了基础。

Objective To clone human Bid gene, express in prokaryotic cells and purify the expressed product. Methods Human Bid gene was amplified from cDNA library of HeLa cells by PCR and cloned into expression vector pGEX-6P-1. The constructed recombinant plasmid was transformed to E. coli BL21 （DE3）for expression under induction of IPTG. The expressed product was purified by GST affinity chromatography. Results The DNA fragment at a length of 588 bp was amplified by PCR. Restriction analysis and sequencing proved that recombinant plasmid pGEX-6P-1-Bid was constructed correctly. The expressed product, with a relative molecular mass of about 48 000, contained about 50% of total somatic protein and reached a purity of 97% after purification. Conclusion Human Bid gene was successfully cloned, and Bid protein with high purity was expressed in prokaryotic cells, which laid a foundation of further study on structure and function of Bid protein.