利用NusA促溶标签原核表达Butelase 1蛋白

Prokaryotic Expression of Protein Butelase 1 through Solubility-enhancing Tag NusA

为获得可溶性Butelase 1蛋白,将Butelase 1基因插入到带有NusA促溶标签的原核表达载体上,将重组质粒转化至大肠杆菌(E.coli)BL21(DE3)感受态细胞中进行诱导表达,用Ni-NTA亲和层析法纯化表达的可溶性蛋白,用SDS-PAGE和Western blot分析蛋白的表达情况。酶切和测序结果证实p ET21b-His6-NusA-Butelase 1原核表达载体构建成功。SDS-PAGE分析结果表明诱导后表达的融合蛋白大小为94.9 k D,主要为可溶性表达,部分为包涵体表达。Western blot检测结果显示,纯化得到的His6-NusA-Butelase 1融合蛋白用抗His6-Tag抗体鉴定时为特异性表达。

In order to obtain soluble protein Butelase 1,we inserted the gene Butelase 1 into the prokaryotic expression vector with solubility-enhancing tag Nus A,transformed the confirmed recombinant plasmids into the competent cells BL21（ DE3） of Escherichia coli to induce their expression,used the method of Ni-NTA affinity chromatography to purify the expressed soluble fusion protein,and utilized SDS-PAGE and Western blot to analyze the prokaryotic expression of this fusion protein. Restriction endonuclease digestion and DNA sequencing analysis proved that the prokaryotic expression vector p ET21b-His6-Nus A-Butelase 1had been constructed successfully. SDS-PAGE analysis indicated that the induced expressive fusion protein was 94.9 k D,and it expressed mainly in the soluble body and partly in the inclusion body. Western blot detection showed that the His6-Tag antibody could specifically recognize the purified His6-Nus A-Butelase 1 fusion protein.