GST-Smad4融合蛋白的表达与纯化
Prokaryotic expression and purification of GST-Smad4 protein
目的：原核表达Smad4基因，纯化获得GST-Smad4融合蛋白。方法：以人表皮HaCaT细胞的cDNA为模板，利用PCR扩增含有BamHI和Sal I 酶切位点的Smad4基因，然后将其克隆到pGEX-4T-1原核表达载体中，将正确的重组载体转入大肠杆菌BL21（DE3），用IPTG诱导表达，再利用MagneGST particles亲和纯化GST-Smad4融合蛋白，最后通过Western blot鉴定此融合蛋白。结果：成功构建pGEX-4T-1-Smad4原核表达载体；30℃条件下，0.2 mmol/L的IPTG能诱导出大量的可溶性GST-Smad4蛋白；经MagneGST particles纯化的GST-Smad4蛋白可被Smad4的抗体特异识别。结论：成功在大肠杆菌中表达了GST-Smad4蛋白，纯化的GST-Smad4蛋白可用于后续的生物学研究。
Objective: To express human Smad4 gene in prokaryotic cells and purify the GST-Smad4 fusion protein. Methods: The full-length Smad4 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1.The recombinant plasmid pGEX-4T-1-Smad4 was transformed into E.coli BL21（DE3）and exogenous protein was induced by IPTG. After purification using MagneGST particles,the GST-Smad4 fusion protein was further identified by Western blot. Results: The recombinant plasmid pGEX-4T-1-Smad4 was constructed successfully. When BL21(DE3) cells transformed with pGEX-4T-1-Smad4 were cultured at 30℃ and induced with 0.2 mmol/L IPTG,GST-Smad4 protein was obtained in a large quantity in supernatant. The purified GST-Smad4 was further identified specifically by Smad4 antibody. Conclusion: GST-Smad4 fusion protein was successfully expressed and prufied, and it could be used for further study of the function of Smad4.
徐志卿、杨予涛
分子生物学生物工程学生物化学
Smad4原核表达蛋白纯化
Smad4prokaryotic expressionprotein purification
徐志卿,杨予涛.GST-Smad4融合蛋白的表达与纯化[EB/OL].(2012-06-04)[2025-05-01].http://www.paper.edu.cn/releasepaper/content/201206-56.点此复制
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