期刊文献+

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
原文传递
导出
摘要 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.
出处 《现代食品科技》 EI CAS 北大核心 2015年第12期128-133,共6页 Modern Food Science and Technology
基金 国家自然科学基金资助项目(21306055) 高等学校博士学科点专项科研基金(20130172120041) 广东省自然科学基金资助项目(2014A030313261)
关键词 乳酸乳球菌 锚定蛋白 绿色荧光蛋白 回向锚定 Bacteria Bioactivity Biological systems Cell membranes DNA Electrophoresis Escherichia coli Fluorescence Fluorescence microscopy Gene expression Polymerase chain reaction Recombinant proteins Sodium dodecyl sulfate Sodium sulfate Strain Temperature
  • 相关文献

参考文献14

  • 1Steen, Anton,Buist, Girbe,Leenhouts, Kees J.,El Khattabi, Mohamed,Grijpstra, Froukje,Zomer, Aldert L.,Venema, Gerard,Kuipers, Oscar P.,Kok, Jan.Cell wall attachment of a widely distributed peptidoglycan binding domain is hindered by cell wall constituents. Journal of Biological Chemistry . 2003
  • 2Maarten L. van Roosmalen,Rolf Kanninga,Mohamed El Khattabi,Jolanda Neef,Sandrine Audouy,Tjibbe Bosma,Anneke Kuipers,Eduard Post,Anton Steen,Jan Kok,Girbe Buist,Oscar P. Kuipers,George Robillard,Kees Leenhouts.??Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria(J)Methods . 2005 (2)
  • 3Jerry Wells.??Mucosal Vaccination and Therapy with Genetically Modified Lactic Acid Bacteria(J)Annual Review of Food Science and Technology . 2011
  • 4余丽芸,王桂华,唐彦君.乳酸菌作为口服疫苗载体的研究进展[J].生物技术通报,2008,24(5):48-50. 被引量:10
  • 5李小华,黄新凤,邵小虎,李林.利用乳酸乳球菌AcmA表面展示β-1,3-1,4-葡聚糖酶[J].生物工程学报,2009,25(1):89-94. 被引量:10
  • 6黄新凤,李小华,李林.利用N-乙酰葡糖胺糖苷酶在乳酸乳球菌表面展示超氧化物歧化酶[J].微生物学通报,2010,37(7):992-998. 被引量:4
  • 7SA Audouy,van Selm S,van Roosmalen ML,E Post,R Kanninga,J Neef,S Esteva?o,EE Nieuwenhuis,PV Adrian,K Leenhouts,PW Hermans.Development of lactococcal GEM-based pneumococcal vaccines. Vaccine . 2007
  • 8Nicola J Hack,Brian Billups,Peter B Guthrie,John H Rogers,Elizabeth M Muir,Thomas N Parks,Stanley B Kater.??Green fluorescent protein as a quantitative tool(J)Journal of Neuroscience Methods . 2000 (2)
  • 9IKAWA M,KOMINAMI K,YOSHIMURA Y,et al.Green fluorescentprotein as a marker in transgenic mice. Development,Growth&Differentiation . 1995
  • 10Chalfie M,Tu Y,Eukivchen G,et al.Green fluorescent protein as a marker for gene expression. Science . 1994

二级参考文献74

  • 1林庆斌,廖升荣,熊亚红,乐学义.超氧化物歧化酶(SOD)的研究和应用进展[J].化学世界,2006,47(6):378-381. 被引量:78
  • 2姚晓英,李大金,袁敏敏.表达hCGβ的重组乳酸杆菌经阴道免疫小鼠后的体液免疫应答[J].中华微生物学和免疫学杂志,2006,26(7):659-664. 被引量:8
  • 3何火聪,刘树滔,潘剑茹,陈躬瑞,饶平凡.GST-TAT-GFP融合蛋白的表达、纯化及鉴定[J].福建医科大学学报,2006,40(4):334-337. 被引量:4
  • 4Buist G, Karsens H, Nauta A, et al. Autolysis of Lactococcus lactis caused by induced overproduction of its major autolysin, AcmA. Appl Environ Microbiol, 1997, 63(7): 2722-2728.
  • 5Buist G, Kok J, Leenhouts KJ, et al. Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. J Bacteriol, 1995, 177: 1554-1563.
  • 6Leenhouts K, Buist G, Kok J. Anchoring of proteins to lactic acid bacteria. Antonie van Leeuwenhoek, 1999, 76: 367-376.
  • 7Ramasamy R, Yasawardena S, Zomer A, et al. Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations. Vaccine, 2006, 24: 3900-3908.
  • 8Tarahomjoo S, Katakura Y, Satoh E, et al. Bidirectional cell-surface anchoring function of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403. J Biosci Bioeng, 2008, 105: 116-121.
  • 9van Roosmalen ML, Kanninga R, E1 Khattabi M, et al. Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria. Methods, 2006, 38: 144-149.
  • 10Okano K, Zhang Q, Kimura S, et al. System using tandem repeats of the cA peptidoglycan-binding domain from Lactococcus lactis for display of both N- and C-terminal fusions on cell surfaces of lactic acid bacteria. Appl Environ Microbiol, 2008, 74: 1117-1123.

共引文献30

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部