UHRF2结构域突变体的原核表达与纯化

Prokaryotic Expression and Purification of Domain Based Mutants of UHRF2

目的构建UHRF2各个以结构域为基础的突变体原核表达载体，在大肠埃希菌中表达并对融合蛋白进行纯化和鉴定。方法以pCMV-3xFlag—UHRF2为模板，PCR扩增UHRF2的各个结构域基因片段，各PCR产物经酶切后连接到pGEX-4T-1载体上；将重组载体转化大肠埃希菌（BL21菌株），IPTG诱导表达各GST融合蛋白，超声波破碎细菌，离心收获蛋白并经谷胱甘肽琼脂糖凝胶4B（glutathione sepharose 4B）亲合纯化；纯化的蛋白经SDS-PAGE电泳后用考马斯亮蓝染色或免疫印记实验鉴定各蛋白表达情况。结果成功构建了UHRF2结构域突变体的原核表达载体，各突变体蛋白表达正确。结论UHRF2各结构域突变体的成功构建便于用GST pull—down实验研究UHRF2参与与其它蛋白相互作用的结构域，为了解UHRF2功能打下了基础。

Objective To construct domain-based GST （glutathione-S-transferase） fusion mu- tant expressing vectors of UHRF2, purify and verify all the prokaryotically expressed proteins for downstream purpose. Methods DNA fragments of each domain of UHRF2 were amplified by PCR method; the PCR products were digested with restriction enzymes and incorporated into pGEX-4T-1 vector. All established mutants were expressed in the transformed BL21 strain of E. coli by IPTG in- duction. The GST-fusion mutants containing bacteria were collected, sonicated and purified for the proteins using Glutathione Sepharose 4B. Purified proteins were subjected to SDS-PAGE followed by CBB staining or immunobloting assay. Results All the domain-based GST fusion mutants of UHRF2 were constructed successfully into prokaryotic expressing vectors and prokaryotically expressed prod- ucts were purified and confirmed. Conclusion The constructed domain-based mutants of UHRF2 can be used in GST pull-down assay to study UHRF2 domains that may be implicated in its partner association, thus enabling further understanding of UHRF2.