摘要
目的探讨人果蝇肿瘤抑制蛋白h Dlg的PDZ结构域在大肠杆菌中的表达与纯化。方法从He La细胞中设计引物克隆含有h Dlg PDZ c DNA片段后进行第二轮扩增,并连接至T载体进行测序鉴定,Eco RⅠ和XhoⅠ双酶切后连接至p GEX-6p-1原核表达载体。转化BL21大肠杆菌后37℃诱导表达。结果测序和双酶切鉴定h Dlg的PDZ结构域被成功克隆至p GEX-6p-1原核表达载体。37℃诱导表达后,大部分GST-PDZ融合蛋白以包涵体的形式存在,部分存在于细菌裂解上清液中。16℃诱导表达后,无法检测到目的蛋白的表达。通过GST-beads结合的方式对细菌裂解液进行纯化,可溶性GST-PDZ的蛋白量可以显著提高。结论通过GST-PDZ诱导表达,再通过GST-beads结合纯化的方式成功表达可溶性的h Hlg PDZ结构域。
Objective To investigate the expression and purification ofhDlg PDZ domain in E.coli BL21 strain. Methods The eDNA fragment containing PDZ domain was cloned from total RNA of HeLa cell. The second round PCR was conducted to clone the PDZ domain. Then the PDZ domain was ligated to T vector. EcoR I and Xho I were used to release the PDZ domain with adequate restriction cites from T vector and the released fragment was cloned to pGEX-6p-1 expressing vector for prokaryotic expression in E.coli BL21. Results DNA sequencing and restriction enzyme digest were conducted to verify the insertion of PDZ domain in pGEX-6p-1 vector. After IPTG induction under 37℃, the GST-fusion protein was mainly existed as inclusion body with less fusion protein existed in supernatant. However, the fusion protein expression was under detectable level if IPTG induction was performed under 16℃. The GST-beads binding assay could be conducted to enrich the GST-PDZ from supernatant of bacterial lysate. Conclusion The PDZ domain of hDlg could be successfully expressed as GST fusion protein and GST-beads binding assay could be used for purify the soluble form of GST-PDZ.
出处
《实用皮肤病学杂志》
2015年第6期415-418,共4页
Journal of Practical Dermatology