β-Ogg1与H-Ras可溶性原核表达纯化及其相互作用的鉴定

Prokaryotic expression, purification and interaction of soluble β-Ogg1 and H-Ras

目的获得外源表达的可溶性β-Ogg1和H-Ras蛋白并鉴定其相互作用。方法将β-Ogg1、H-Ras基因构建到含有GST标签的p GEx-4T-2-TEV和6*His标签的p ET-28a-TEV原核表达载体上,转化至大肠杆菌BL21(DE3)。采用SDSPAGE检测表达产物,GST介导的Pulldown方法鉴定蛋白相互作用。结果成功构建了β-Ogg1和H-Ras基因原核表达载体并转化大肠杆菌。在8-oxo G小分子作用下,β-Ogg1与H-Ras有直接相互作用。结论采用原核表达和亲和层析纯化得到可溶性GST_β-Ogg1(25-424)和6*His_H-Ras融合蛋白,并初步证实β-Ogg1和H-Ras存在直接相互作用。

Objective To obtain the in vitro expression and interaction of soluble β-Ogg1 and H-Ras. Methods Both β-Ogg1 and H-Ras genes were constructed into the prokaryotic expression vectors p GEx-4T-2-TEV with GST tag and p ET-28 aTEV with 6＊His tag, followed by transfection with Escherichia coli BL21（DE3）, and the target recombinant proteins were induced with IPTG. The expression products and protein interaction were detected using SDS-PAGE and GST-mediated Pulldown assay. Results The prokaryotic expression vectors of β-Ogg1 and H-Ras were successfully constructed and transformed into DE3. There was a direct interaction between Ogg1 and H-Ras in the presence of 8-oxo G. Conclusion The soluble GSTβ-Ogg1（25-424） and 6＊HisH-Ras fusion proteins are purified using prokaryotic expression and affinity chromatography. A direct interaction between β-Ogg1 and H-Ras is present.