利用GST融合蛋白表达系统表达与纯化人N-Ras蛋白

摘要

Objective: To construct the prokaryotic expression vector of human N-Ras gene, obtain the purified GST-N-Ras protein. Methods: Human N-Ras gene coding region was amplified from human mammary DNA li⁃brary by the technique of PCR, and was inserted into prokaryotic expression vector pGEX-KG. The recombinant plasmid pGEX-KG-N-Ras was transformed into E.coli Rossate. The expressed product was purified by GST-Sepha⁃rose 4B beads and identified by SDS-PAGE analysis. Results: The DNA fragment of N-Ras about 600 bp was amplified by PCR, successfully cloned into pGEX-KG and identified by sequencing. The recombinant protein GST-N-Ras with Mr 47 kD was successfully induced, purified well. Conclusion: The recombinant protein of GST-N-Ras is obtained successfully, which lay the foundation for further research between Ras gene and other gene, in⁃cluding oncogene and tumor suppressor gene.%目的：克隆人N-ras蛋白全长编码区基因，获得其原核表达产物，并对融合蛋白进行纯化。方法：采用PCR技术从人乳腺文库中扩增出人N-ras蛋白全长编码区基因，将其克隆到pGEX-KG载体中，在大肠杆菌Rossate中表达后，利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化，SDS-PAGE鉴定表达与纯化产物。结果：从人乳腺文库中扩增获得约600 bp的DNA片段，并克隆至pGEX-KG载体上，经测序与目的序列完全一致；在大肠杆菌Rossate中诱导表达出相对分子质量约47×103的目的蛋白；纯化后，经鉴定获得了纯度较高的重组蛋白GST-N-Ras。结论：获得了重组蛋白GST-N-ras，为后续深入研究Ras基因与其他癌基因、抑癌基因的相互作用奠定了基础。