利用原核表达系统一步法制备生物素标记蛋白质

doi:10.13523/j.cb.20140106

中国生物工程杂志

2014, Vol. 34

Issue (1): 36-41 DOI: 10.13523/j.cb.20140106

技术与方法

利用原核表达系统一步法制备生物素标记蛋白质

谢小丽¹, 程剑松¹, 王晓敏¹, 梅香寒¹, 刘琳¹, 李静^1,2

1. 南开大学药学院天津市分子药物研究重点实验室天津 300071;
2. 天津化学化工协同创新中心天津 300071

One-Step Preparation of Biotinylated Protein Using Prokaryotic Expression System

XIE Xiao-li¹, CHENG Jian-song¹, WANG Xiao-min¹, MEI Xiang-han¹, LIU Lin¹, LI Jing^1,2

1. College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China;
2. Synergetic Innovation Center of Chemical Science and Engineering, Tianjin 300071, China

全文: PDF(672 KB) HTML

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80 μmol/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。

关键词： 原核表达; 生物素标记; 己糖苷酶D

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 co-expression 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 co-expressed 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.

Key words: Prokaryotic expression Biotinylation Hexosaminidase D

收稿日期: 2013-10-12 出版日期: 2014-01-25

ZTFLH:

Q563

基金资助: 国家自然科学基金资助项目（91013013，31000371）

通讯作者: 李静，E-mail：jinglink@nankai.edu.cn E-mail: jinglink@nankai.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	谢小丽
	程剑松
	王晓敏
	梅香寒
	刘琳
	李静

引用本文:

谢小丽, 程剑松, 王晓敏, 梅香寒, 刘琳, 李静. 利用原核表达系统一步法制备生物素标记蛋白质[J]. 中国生物工程杂志, 2014, 34(1): 36-41.

XIE Xiao-li, CHENG Jian-song, WANG Xiao-min, MEI Xiang-han, LIU Lin, LI Jing. One-Step Preparation of Biotinylated Protein Using Prokaryotic Expression System. China Biotechnology, 2014, 34(1): 36-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140106 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I1/36

[1] Green N M. Avidin and streptavidin. Methods in Enzymol, 1990, 184: 51-67.
[2] Hatakeyama K, Tanaka T, Sawaguchi M, et al. Microfluidic device using chemiluminescence and a DNA-arrayed thin film transistor photosensor for single nucleotide polymorphism genotyping of PCR amplications from whole blood. Lab Chip, 2009, 9: 1052-1058.
[3] Howarth M, Takao K, Hayashi Y, et al. Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc Nat Acad Sci US, 2005, 102(21): 7583-7588.
[4] Larsson C, Rodahl M, Höök F. Characterization of DNA immobilization and subsequent hybridization on a 2D arrangement of streptavidin on a biotin-modified lipid bilayer supported on SiO₂. Anal Chem, 2003, 75: 5080-5087.
[5] Matsunaga T, Maeda Y, Yoshino T, et al. Fully automated immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micropolystyrene bead composites,‘Beads on Beads’. Anal Chem Acta, 2007, 597: 331-339.
[6] 路静, 施前. 生物素标记法分析MCF-10A细胞质膜蛋白质. 复旦学报(医学版), 2010, 37(6): 710-714. Lu J, Shi Q. Analysis on plasma membrane proteins of MCF10A by cell surface biotinylation. Fudan Univ J Med Sci, 2010, 37(6): 710-714.
[7] Bayer E A, Wilchek M. Protein biotinylation. Methods in Enzymol, 1990, 184: 138-160.
[8] Fall R R. Analysis of microbial biotin protein. Methods in Enzymol, 1979, 62: 390-398.
[9] Barker D F, Campbell A M. Genetic and biochemical characterization of the birA gene and its product:Evidence for a direct role of biotin holoenzyme sythetase in repression of the biotin operon in Escherichia coli. J Mol Biol, 1981, 146(4): 469-492.
[10] Li Y F, Sousa R. Expression and purification of E.coli BirA biotin ligase for in vitro biotinylation. Prot Expr Purif, 2012, 82: 162-167.
[11] Dorothy B, Kovaleva E, Schatz P J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci, 1999, 8: 921-929.
[12] Gutternigg M, Rendic D, Voglauer R, et al. Mammalian cells contain a second nucleocytoplasmic hexosaminidase. Biochem J, 2009, 419(1): 83-90.
[13] 刘琳, 徐, 蔡春梅, 等. 人己糖胺酶D的原核表达, 纯化及酶学特性研究. 中国生物工程杂志, 2012, 32(9): 28-33. Liu L, Xu Y, Cai CH M, et al. Expression, purification and enzymatic characteristics of human Hex D in E.coli. China Biotechnology, 2012, 32(9): 28-33.
[14] Romier C, Jelloul M B, Albeck S, et al. Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallographica Section D: Biological Crystallography, 2006, 62(10): 1232-1242.
[15] Choi R E, Schulman H, Cronan J E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Sci, 2004, 13(11): 3043-3050.

[1]	乔圣泰,王曼琦,徐慧妮. 番茄SlTpx原核表达蛋白的体外功能分析^*[J]. 中国生物工程杂志, 2021, 41(8): 25-32.
[2]	张磊,唐永凯,李红霞,李建林,徐逾鑫,李迎宾,俞菊华. 促进原核表达蛋白可溶性的研究进展 ^*[J]. 中国生物工程杂志, 2021, 41(2/3): 138-149.
[3]	张潇航,李媛媛,贾敏晅,顾奇. 弹性蛋白样生物材料的制备及性质鉴定 ^*[J]. 中国生物工程杂志, 2020, 40(8): 33-40.
[4]	吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. **LbCpf1基因的原核表达、纯化与体外切割检测 ^***[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[5]	李彤彤,宋彩玲,杨凯越,王文静,陈慧宇,刘明. 抗犬细小病毒VP2蛋白单链抗体的制备与中和活性研究 ^*[J]. 中国生物工程杂志, 2020, 40(4): 10-16.
[6]	陈秋利,杨丽超,李辉,温莎,李刚,何敏. 人Nek2蛋白原核表达纯化及其多克隆抗体制备 ^*[J]. 中国生物工程杂志, 2020, 40(3): 31-37.
[7]	杨隆兵,国果,马慧玲,李妍,赵欣宇,苏佩佩,张勇. 家蝇抗菌肽AMPs17蛋白原核表达条件的优化及其抗真菌活性检测 ^*[J]. 中国生物工程杂志, 2019, 39(4): 24-31.
[8]	李明英,王仁军,张帆,迟彦. β2糖蛋白Ⅰ第五结构域及其突变体、短肽片段的原核表达及活性分析 ^*[J]. 中国生物工程杂志, 2018, 38(8): 1-9.
[9]	陈远侨,龙定沛,豆晓雪,祁润,赵爱春. ELP₃₀-tag蛋白纯化能力的原核表达研究[J]. 中国生物工程杂志, 2018, 38(2): 54-60.
[10]	何亚南,孙钰椋,任雅坤,梁盛英,杨芬,刘彦礼,林俊堂. 金黄色葡萄球菌类肠毒素K与GFP融合蛋白工程菌的构建及其表达蛋白生物学活性分析 ^*[J]. 中国生物工程杂志, 2018, 38(12): 14-20.
[11]	任建委,李军,李尚泽. 人源CT55蛋白原核表达及单克隆抗体的制备 ^*[J]. 中国生物工程杂志, 2018, 38(11): 1-8.
[12]	孙文佳, 姚宇峰, 杨旭, 黄惟巍, 刘存宝, 龙琼, 褚晓杰, 马雁冰. 乙肝核心抗原病毒样颗粒呈现HPV 16L1抗原表位及特异抗体诱导[J]. 中国生物工程杂志, 2017, 37(3): 58-64.
[13]	祖力皮也·吐尔逊, 曹春宝, 温浩, 丁剑冰, 德力夏提·依米提. 细粒棘球蚴EgG1Y162基因进化分析、表达及鉴定[J]. 中国生物工程杂志, 2016, 36(4): 78-87.
[14]	周亮, 叶浩, 周瓅, 关文, 李京敬, 郜尽, 韩伟, 俞雁. 人CXCL4蛋白原核表达与纯化[J]. 中国生物工程杂志, 2016, 36(1): 7-13.
[15]	黄健, 黄美容, 朱杰华, 骆诗露, 闵迅. 肺炎链球菌SP0306蛋白的表达纯化及结晶研究[J]. 中国生物工程杂志, 2015, 35(6): 21-25.

Viewed

Full text

Abstract

Cited

Shared

Discussed