分选酶A(SrtA)的GST融合表达、纯化及活性测定  

GST Fusion Expression,Purification and Activity Assay of SortaseA

作　　者：邓思[1] 罗立新[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广州510006

出　　处：《生物技术通报》2012年第5期121-125,共5页Biotechnology Bulletin

摘　　要：构建GST/金黄色葡萄球菌分选酶A(SrtA)的原核表达载体,在大肠杆菌中表达、纯化分选酶,并利用展示在酵母表面的底物检测分选酶的活性。以pMD20-SrtA为模板,PCR扩增得到SrtA△N59基因,经BamHⅠ和XhoⅠ双酶切,连接到原核表达载体pGEX-4T-1中,构建重组表达载体pGEX-SrtA△N59,转化大肠杆菌BL21(DE3),IPTG诱导表达,GST亲和层析分离纯化得到SrtA△N59,与展示在酵母表面的底物序列QALPETGEE-linker-EGFP作用,产生游离的EGFP,通过酶标仪检测EGFP荧光强度确定分选酶的活性。结果显示,重组表达载体pGEX-SrtA△N59经IPTG诱导,表达出相对分子质量约为42 kD的融合蛋白,SDS-PAGE分析,该融合蛋白是以可溶形式表达。分离纯化得到的分选酶与底物作用,其荧光强度由568.66±12.14增加至921.43±13.02。以上结果表明,成功构建了重组表达载体pGEX-SrtA△N59,并在大肠杆菌中获得了可溶表达的有活性的分选酶。It was to clone and express the Staphylococcus aureus sortaseA （ SrtA ） gene in prokaryotic expression system and to purify the GST fused protein. Then the activity of SrtA was detected by the substrate displayed on yeast cell surface. The truncated SrtA gene, SrtA △N59, was amplified by PCR using pMD20-SrtA as the template. The SrtA△ N59 gone was digested with BamH I and Xho I , and inserted into vector pGEX-4T-1. Then, the recombinant expression vector pGEX-SrtAaN59 was transformed into Escherichia coli BL21 （ DE3 ） and induced with IPTG. The protein SrtA△N59 was purified by GST affinity chromatography. The activity of SrtA△N59 was detected by the fluorescence intensity of free EGFP generated from the interaction of sortase with its substrate displayed on yeast surface. The SDS-PAGE result showed that approximately 42 kD protein was expressed by pGEX-SrtA△N59. The fluorescence intensity of free EGFP increased from 568.66 ± 12.14 to 921.43 ± 13.02 after interaction of sortase with its substrate. These results suggested that the recombinant expression vector pGEX-SrtA△N59 was successfully constructed and the active SrtA was soluble when expressed in Escherichia coli.

关 键 词：分选酶A GST融合表达 纯化 底物 活性测定 

分 类 号：Q78[生物学—分子生物学]