cA-GFP融合蛋白在大肠杆菌中的高效功能性表达

Efficient and Functional Expression of cA-GFP Fusion Protein in Escherichia coli

为了实现乳酸乳球菌锚定蛋白cA在大肠杆菌中可溶表达以及直观检测其生物学活性,以乳酸乳球菌MG1363为模板,扩增N-乙酰葡萄糖胺糖苷酶AcmA基因,将其C末端序列与GFP基因融合,连接至大肠杆菌表达载体pET28a,构建重组质粒pET28a-cA-GFP,转化至大肠杆菌表达菌株BL21(DE3),通过低温诱导表达重组蛋白cA-GFP。工程菌超声破碎后的上清液与经热酸处理的乳酸乳球菌常温孵育,经SDS-PAGE和荧光显微镜检测。结果表明,工程菌可以表达分子量约53ku的目的融合蛋白质,与预期大小相符。cA-GFP通过锚定蛋白cA的引导回向锚定,成功将GFP展示在乳酸乳球菌表面,目的蛋白cA-GFP在乳酸乳球菌表面展示量为121mg/g干重菌体。GFP锚定至乳酸乳球菌后于4℃保存,连续6d测定其荧光强度,荧光强度仍可达82.2%,证明其稳定性较好。成功获得了具有生物学功能的cA-GFP可溶性蛋白,为进一步展示功能性外源蛋白奠定了坚实的基础。

To achieve soluble expression of the Lactococcus lactis anchoring protein cA (C-terminal region of N-acetylmuraminidase [AcmA]) in Escherichia coli, and elucidate its biological activity, the AcmA gene was amplified by polymerase chain reaction (PCR) using L. lactis MG1363 as the template. The C-terminal of AcmA was fused with green fluorescent protein (GFP), followed by ligation of the chimeric cA-GFP into the E. coli expression vector pET28a, yielding the recombinant plasmid pET28a-cA-GFP. Subsequently, the recombinant plasmid was transformed into E. coli strain BL21 (DE3), and expression of the recombinant cA-GFP protein was induced at low temperature. After ultrasonic disruption of the recombinant strains, the supernatant was directly incubated with acid-pretreated L. lactis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence microscopy analyses were conducted. The results showed that the cA-GFP fusion protein with a molecular weight (MW) of about 53 ku (as expected) was expressed in the engineered bacteria. cA-GFP was successfully docked onto the cell surface of L. lactis via the inverse docking directed by the anchoring protein cA. One gram of dry bacteria could load 121 mg of cA-GFP protein. The fusion protein (cA-GFP) was very stable and the fluorescence intensity could be maintained at 82.2% when the proteins were stored at 4 for six days. The cA-GFP soluble protein with biological function was successfully obtained, which provides a solid basis for developing exogenous proteins with biological functions. © 2015, South China University of Technology. All right reserved.