转录因子LRF蛋白POZ结构域的表达与纯化

Prokaryotic expression and purification of POZ domain of rat LRF protein

[目的]旨在原核表达转录因子LRF的POZ结构域,纯化获得GST-POZ融合蛋白。[方法]以SD大鼠海马组织c DNA为模板,利用PCR扩增带有EcoRⅠ和XhoⅠ酶切位点的LRF基因的POZ结构域,并将其插入到原核表达载体p GEX-4T-1中,将构建成功的p GEX-4T-1-POZ原核表达质粒转化到大肠杆菌BL21(DE3),用IPTG诱导融合蛋白表达,再利用MagneGST particles亲和纯化GST-POZ融合蛋白,最后通过Western Blot鉴定融合蛋白。[结果]p GEX-4T-1-POZ原核表达质粒构建成功;在37℃条件下,浓度为0. 2 mmol/L的IPTG诱导7 h能够使重组蛋白大量表达,经MagneGST particles纯化后的GST-POZ重组蛋白能够被识别LRF的抗体特异性识别。[结论]纯化后的GST–POZ重组蛋白能够用于后续的生物学研究。

[Objective] To express the POZ domain of rat LRF gene in prokaryotic cells and purify the GST-POZ fusion protein.[Method]The POZ domain of LRF gene was amplified by PCR and inserted into prokaryotic expression vector. The recombinant p GEX-4 T-1-POZ plasmid was transformed into E. coli BL21( DE3) and exogenous protein was induced by IPTG.After purification using MagneGST particles,the GST-POZ fusion protein was further identified by Western Blot analysis. [Result] The p GEX-4 T-1-POZ prokaryotic expression vector was successfully constructed. The optimal concentration of IPTG was 0. 2 mmol/L 7 hours at 37 ℃ . The purified GST-POZ fusion protein could be specifically recognized by LRF antibody.[Conclusion]The GST-POZ fusion protein was successfully expressed and purified. GST-POZ fusion protein can be used for further biological research.