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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 of PDZ domain of human Drosophila tumor suppressor protein hDlg in Escherichia coli. METHODS: Primers of HeLa cells were designed and cloned into a DNA fragment containing hDlg PDZc, followed by a second round of amplification and ligated to the T vector for sequencing. The double digested EcoRI and XhoI were ligated into pGEX-6p-1 pronuclei Expression vector. After transformed into E. coli BL21, the expression was induced at 37 ℃. Results The PDZ domain of hDlg was successfully cloned into pGEX-6p-1 prokaryotic expression vector by sequencing and double enzyme digestion. After induction at 37 ° C, most of the GST-PDZ fusion proteins were present as inclusion bodies, partially in the bacterial lysis supernatant. After induction at 16 ℃, the expression of the target protein could not be detected. Purification of the bacterial lysate by GST-beads incorporation can significantly increase the amount of protein in soluble GST-PDZ. Conclusion The expression of soluble hHlg PDZ domain was successfully induced by GST-PDZ and then purified by GST-beads binding.