【摘要】 传统的蛋白质生物素标记多采用体外化学修饰法,涉及生物素和蛋白质的活化、透析和纯化等多种处理,该方法步骤繁琐,且对目的蛋白的损失较大。本实验利用原核共表达质粒pCDFDuet-1,将含有6个组氨酸标签的人己糖苷酶D(hexosaminidase D,HexD)的cDNA与生物素受体多肽(biotin acceptor peptide,BAP)DNA进行PCR拼接,连入pCDFDuet-1的多克隆位点1(multiple cloning site1,MCS1);将以大肠杆菌Trans5α基因组为模板克隆得到的生物素连接酶(biotin ligase,BirA)基因连入MCS2,构建重组质粒pCDFDuet-hexD-BAP-birA。初步验证后将该质粒转化至大肠杆菌BL21(DE3)pLysS中,利用0.1 mmol/L的IPTG和80μmol/L的生物素进行诱导表达,采用Ni-NTA亲和层析和超滤对HexD进行纯化,SDS-PAGE检测分子量的大小(60 kDa)和纯度(90%以上)。以anti-HexD和链霉亲和素-HRP为抗体,Western blot检测发现,HexD-BAP表达正确,且被生物素标记;同时以4-MU-O-GalNAc为荧光底物,检测到生物素化标记HexD的糖苷酶活性为3.6 nmol/(min·μg),与未标记HexD的活性(3.06 nmol/(min·μg))相当。结果表明,可以利用BirA及其受体多肽,通过共表达质粒pCDFDuet-1,一步转化、表达和纯化,在大肠杆菌中进行外源蛋白的表达和生物素标记,且不改变目的蛋白的生物活性,可应用于免疫标记、互作蛋白的捕获等生物学研究。更多还原

【Abstract】 Traditionally,proteins were labeled with biotin in vitro via chemical modification which involved in chemical activation,dialysis and purification of biotin and target protein. This process was exhausting,and often required excessive target protein for labelling. In this study,using pCDFDuet-1 as a prokaryotic coexpression plasmid,the cDNA of hexD( Hexosaminidase D) containing 6x His and the DNA of BAP( biotin acceptor peptides) were fused by PCR and inserted into MCS1( Multiple cloning site1). birA( Biotin ligase) gene cloned from E. coli Trans5α genome was inserted into MCS2. The constructed recombinant plasmid pCDFDuet-hexD-BAP-birA was then verified by sequencing and transformed into E. coli BL21( DE3) pLysS. The cell was induced by adding 0. 1 mmol / L IPTG and 80 μmol / L biotin,and HexD-BAP was expressed and purified via Ni-NTA affinity chromatography and ultrafiltration. The molecular weight( 60 kDa) and the purity( > 90%) of the protein were verified via SDS-PAGE. Using anti-HexD and streptavidin-HRP as antibodies, western blots showed that the recombinant HexD-BAP was expressed and successfully labeled with biotin by coexpressed BirA. By using 4-MU-O-GalNAc( 4-Methylumbelliferyl 2-acetamido-2-deoxy-β-D-galctopyranoside) as substrate,the activity of biotin labeled HexD-BAP was determined as 3. 6 nmol /( min·μg),which has a good match of the activity of the unlabeled HexD( 3. 06 nmol /( min·μg)). These results demonstrated that via BirA and BAP co-expression,exogenous proteins could be expressed and labeled with biotin in vivo without altering their bioactivity. This system involves one-step transformation,expression and purification,which can be an efficient and useful tool for many biological studies such as immune-labeling and interactive protein trap.更多还原