GST-FHL2融合蛋白的表达及纯化

Expression and purification of GST-FHL2 fusion protein

目的高效表达和纯化可溶性GST-FHL2融合蛋白。方法(1)PCR法扩增FHL2 (Four and a half LIM domains 2)基因的编码片段,分别在5'端和3'端加上EcoR I和Xho l酶切位点,并克隆进入原核表达载体pGEX-4T-1;(2)利用异丙基硫代-β-D-半乳糖苷(IPTG)诱导重组质粒pGEX-4T-1-FHL2在大肠杆菌B121(DE3)中表达同时带有谷胱甘肽-S-转移酶(GST)标签的融合蛋白;(3)超声法裂解大肠杆菌,应用谷胱苷肽琼脂糖树脂纯化可溶的GST-FHL2融合蛋白;(4)通过SDS-PAGE和Western blot验证GST-FHL2的表达。结果(1)成功构建pGEX-4T-1-FHL2重组质粒,测序结果证明FHL2与载体的GST在同一读框;(2)0.1 mmol/L的IPTG在23℃的条件下能诱导可溶性GST-FHL2融合蛋白高效表达;(3)在Western blot分析中,GST-FHL2能被鼠抗GST单克隆抗体特异性识别,条带所在位置和GST-FHL2的分子量相符。结论正确构建pGEX-4T-1-FHL2重组质粒,在大肠杆菌BL21中高效表达GST-FHL2融合蛋白,经谷胱苷肽琼脂糖树脂纯化得到高纯度的可溶性GST-FHL2融合蛋白。

Objective To highly express and purify the soluble GST-FHL2 fusion protein. Methods （1）The open reading frame of FHL2 was amplified by polymerase chain reaction （PCR）, then, the PCR product was cloned into EcoR Ⅰ/Xho 1 sites in pGEX-4T-1 to construct pGEX-4T-1-FHL2 recombinant plamsid; （2） pGEX-4T-1-FHL2 was transformed into E. eoli BL21 （DE3） and induced with isopropyl-β-D-thiogalactoside （IPTG） to express GST-FHL2 fusion protein; （3） Bacterial ceils were disrupted by sonication, and the soluble fraction of fusion proteins were purified by GST Resin; （4） GST-FHL2 fusion protein was verified by SDS-PAGE and Western blotting anlysis. Results （ 1 ） The recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; （2）At 23℃, soluble GST-FHL2 fusion protein was highly expressed after induced with 0. 1 mM IPTG; （3）GST-FHL2 can be detected by Western blotting using the mouse monoclonal anti-GST antibody. Conclusion The pGEX- 4T-1-FHL2 recombinant plasmid was correctly constructed , GST-FHL2 fusion protein was induced to highly express in BL21, and the pure soluble fusion protein was obtained by purification using GST Resin.