金黄色葡萄球菌脱水鲨烯脱氢酶(CrtN)和DNA结合蛋白Sso7d的融合表达

Fusion Expression of Dehydrosqualene Desaturase(CrtN)from Staphylococcus aureus and DNA Binding Protein Sso7d

分子质量相对小的DNA结合蛋白Sso7d可以帮助蛋白折叠,提高蛋白表达量并有助于晶体生长.为解决金黄色葡萄球菌(Staphylococcus aureus)脱水鲨烯脱氢酶(dehydrosqualene desaturase,Crt N)蛋白较难在大肠杆菌(Escherichia coli)中进行可溶表达的问题,借助小分子DNA结合蛋白Sso7d,将其与Crt N基因通过重叠PCR(Overlapping PCR)连接起来,连至p ET-46 Ek/LIC载体上,在大肠杆菌BL21(DE3)中进行带有6-His标签的融合表达.通过核酸电泳及序列测定进而确定所连接基因的正确性,经Ni-NTA亲和层析与凝胶过滤层析对目的蛋白进行纯化,利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blot)对目的蛋白进行确证.通过对Crt N-Sso7d以及Sso7d-Crt N进行分析比较,发现Crt N-Sso7d的表达量较高且能得到较多高纯度蛋白,这将被用来进行后续蛋白结晶和结构解析工作.

Small molecular-weight DNA binding protein Sso7 d can help protein folding and growing into protein crystals.To obtain dehydrosqualene desaturase（Crt N）protein from Staphylococcus aureus with high purity,Sso7 d was used to help the expression and purification.Sso7 d were ligated on both N and C terminals of Crt N through overlapping polymerase chain reaction（PCR）.After being cloned onto p ET-46 Ek/LIC vector,the eligible plasmids were transformed into E.coli BL21（DE3）for expression after sequencing.IPTG was used to induce the expression of recombinant protein with Histag.The target protein was purified with Ni-NTA affinity chromatography,and gel filtration chromatography and then verified by SDS-PAGE and Western blot.Crt N-Sso7 d showed a better expression level and produced more and better proteins compared with Sso7 d-Crt N.This study can be used for further protein crystallization and structural analysis.