Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2017, Vol. 37 Issue (1): 53-57    DOI: 10.13523/j.cb.20170108
技术与方法     
α-溶血素的表达及其纳米孔的制备
卓丽霞1, 王莹2, 张春萍2, 段静2, 张亚妮1
1. 西北大学生命科学学院 西安 710069;
2. 西北大学化学与材料科学学院 西安 710069
The Expression of α-Hemolysin and the Preparation of Its Nanopore
ZHUO Li-xia1, WANG Ying2, ZHANG Chun-ping2, DUAN Jing2, ZHANG Ya-ni1
1. The College of Life Science, Northwest University, Xi'an 710069, China;
2. College of Chemistry & Mateirals Secience, Northwest University, Xi'an 710069, China
 全文: PDF(574 KB)   HTML
摘要:

α-溶血素(α-hemolysin,αHL)是金黄色葡萄球菌分泌的一种水溶性穿孔毒素,在双脂膜上可组装成结构稳定的七聚体蛋白纳米孔,是理想的单分子检测器件。以大肠杆菌BL21(DE3)pLysS作为表达宿主菌株诱导表达野生型和单位点突变型α-溶血素,超滤膜分子截留法纯化蛋白质,使用兔血红细胞膜将αHL单体组装成七聚体蛋白纳米孔,并在人工脂双层膜上构建适于单分子分析的分子器件。通过溶血实验和单通道电流记录,证实αHL溶血活性良好,组装的αHL七聚体纳米孔在双脂膜上打孔能力和离子通透性良好,结构稳定,可作为理想的单分子检测器件。此方法为大量制备αHL单体及其在单分子分析和应用研究方面奠定了基础。

关键词: α-溶血素单通道电流单分子检测器件蛋白纳米孔    
Abstract:

α-hemolysin (αHL) is a channel-forming toxin released by Staphylococcus aureus, which can form a transmembrane heptamers nanopore on eukaryotic cell membranes and other lipid bilayers. The αHL nanopore has been popularly used as a single molecule biosensor. In the present study, αHL was expressed in the host strain BL21(DE3)pLysS and purified by using molecular weight cut-off of ultrafiltration membranes. αHL heptameric nanopore was formed on rabbit red blood cell memberanes and characterized by using patch clamp technique and single channel current recording. αHL heptameric nanopore showed a stable structure and good ion permeability on lipid bilayer. It can be used as a stable single-molecule detection device. These results make a useful work for the preparation of α-hemolysin heptameric nanopore and the further exploring in the filed of single-molecule detection.

Key words: Single-channel current    Protein nanopore    α-hemolysin    Single-molecule detection device
收稿日期: 2016-08-23 出版日期: 2017-01-25
ZTFLH:  Q819  
基金资助:

国家自然科学基金(21175105),中国博士后基金(2012M521795)资助项目

通讯作者: 张亚妮     E-mail: yani@nwu.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  

引用本文:

卓丽霞, 王莹, 张春萍, 段静, 张亚妮. α-溶血素的表达及其纳米孔的制备[J]. 中国生物工程杂志, 2017, 37(1): 53-57.

ZHUO Li-xia, WANG Ying, ZHANG Chun-ping, DUAN Jing, ZHANG Ya-ni. The Expression of α-Hemolysin and the Preparation of Its Nanopore. China Biotechnology, 2017, 37(1): 53-57.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170108        https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I1/53

[1] Gray G S, Kehoe M. Primary sequence of the alpha-toxin gene from Staphylococcus aureus wood 46. Infection and Immunity, 1984, 46(2):615-618.
[2] Tobkes N, Wallace B A, Bayley H. Secondary structure and assembly mechanism of an oligomeric channel protein. Biochemistry, 1985, 24(8):1915-1920.
[3] Song L, Hobaugh M R, Shustak C, et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science, 1996, 274(5294):1859-1866.
[4] Branton D, Deamer D W, Marziali A, et al. The potential and challenges of nanopore sequencing. Nat Biotechnol, 2008, 26(10):1146-1153.
[5] Kang X F, Cheley S, Rice-Ficht A C, et al. A storable encapsulated bilayer chip containing a single protein nanopore. J Am Chem Soc, 2007, 129(15):4701-4705.
[6] Ayub M, Bayley H. Individual RNA base recognition in immobilized oligonucleotides using a protein nanopore. Nano Lett, 2012, 12(11):5637-5643.
[7] Bond P J, Guy A T, Heron A J, et al. Molecular dynamics simulations of DNA within a nanopore:arginine-phosphate tethering and a binding/sliding mechanism for translocation. Biochemistry, 2010, 50(18):3777-3783.
[8] Cracknell J A, Japrung D, Bayley H. Translocating kilobase RNA through the staphylococcal alpha-hemolysin nanopore. Nano Lett, 2013, 13(6):2500-2505.
[9] Yao F, Duan J, Wang Y, et al. Nanopore single-molecule analysis of DNA-doxorubicin interactions. Anal Chem, 2015, 87(1):338-342.
[10] Cheley S, Braha O, Lu X, et al. A functional protein pore with a "retro" transmembrane domain. Protein Sci, 1999, 8(6):1257-1267.
[11] Japrung D, Henricus M, Li Q, et al. Urea facilitates the translocation of single-stranded DNA and RNA through the alpha-hemolysin nanopore. Biophys J, 2010, 98(9):1856-1863.
[12] 王多宁, 赵雁武, 田芙蓉. 考马斯亮蓝微盘比色法测定蛋白质含量. 第四军医大学学报, 2001, 22(6):528-529. Wang D N, Zhao Y W, Tian F R. Protein quantif ication with coomassie bull iant blue microplate-colormetric. J Fourth Mil Med Univ, 2001, 22(6):528-529.
[13] Rotem D, Jayasinghe L, Salichou M, et al. Protein detection by nanopores equipped with aptamers. J Am Chem Soc, 2012, 134(5):2781-2787.
[14] Bayley H. Designed membrane channels and pores. Curr Opin Biotechnol, 1999, 10(1):94-103.
[15] Gu L Q, Dalla Serra M, Vincent J B, et al. Reversal of charge selectivity in transmembrane protein pores by using noncovalent molecular adapters. Proc Natl Acad Sci USA, 2000, 97(8):3959-3964.
[16] Boersma A J, Brain K L, Bayley H. Real-time stochastic detection of multiple neurotransmitters with a protein nanopore. ACS Nano, 2012, 6(6):5304-5308.
[17] Gu L Q, Cheley S, Bayley H. Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores. J Gen Physiol, 2001, 118(5):481-494.
[18] Hammerstein A F, Jayasinghe L, Bayley H. Subunit dimers of alpha-hemolysin expand the engineering toolbox for protein nanopores. J Biol Chem, 2011, 286(16):14324-14334.
[19] Mantri S, Sapra K T, Cheley S, et al. An engineered dimeric protein pore that spans adjacent lipid bilayers. Nature Communications, 2013, 4(2):216-219.

[1] 林敏. 玉米生物育种基础研究与关键技术[J]. 中国生物工程杂志, 2021, 41(12): 1-3.
[2] 吴函蓉,王莹,黄英明,李冬雪,李治非,方子寒,范玲. 以基地平台为抓手,促进生物技术创新与转化[J]. 中国生物工程杂志, 2021, 41(12): 141-147.
[3] 尹泽超,王晓芳,龙艳,董振营,万向元. 玉米穗腐病抗性鉴定、遗传分析与分子机制*[J]. 中国生物工程杂志, 2021, 41(12): 103-115.
[4] 冷燕,孙康泰,刘倩倩,蒲阿庆,李翔,万向元,魏珣. 全球基因编辑作物监管趋势研究[J]. 中国生物工程杂志, 2021, 41(12): 24-29.
[5] 何伟,祝蕾,刘欣泽,安学丽,万向元. 玉米遗传转化与商业化转基因玉米开发*[J]. 中国生物工程杂志, 2021, 41(12): 13-23.
[6] 杨梦冰,江易林,祝蕾,安学丽,万向元. CRISPR/Cas植物基因组编辑技术及其在玉米中的应用*[J]. 中国生物工程杂志, 2021, 41(12): 4-12.
[7] 殷芳冰,王成,龙艳,董振营,万向元. 玉米雌穗性状遗传分析与形成机制*[J]. 中国生物工程杂志, 2021, 41(12): 30-46.
[8] 秦文萱,刘鑫,龙艳,董振营,万向元. 玉米叶夹角形成的遗传基础与分子机制解析*[J]. 中国生物工程杂志, 2021, 41(12): 74-87.
[9] 王锐璞,董振营,高悦欣,龙艳,万向元. 玉米籽粒淀粉含量遗传基础与调控机制*[J]. 中国生物工程杂志, 2021, 41(12): 47-60.
[10] 马雅杰,高悦欣,李依萍,龙艳,董振营,万向元. 玉米株高和穗位高的遗传基础与分子机制*[J]. 中国生物工程杂志, 2021, 41(12): 61-73.
[11] 王彦博,魏佳,龙艳,董振营,万向元. 玉米雄穗性状遗传结构与形成分子机制*[J]. 中国生物工程杂志, 2021, 41(12): 88-102.
[12] 毛开云,李荣,李丹丹,赵若春,范月蕾,江洪波. 全球双特异性抗体药物研发格局分析*[J]. 中国生物工程杂志, 2021, 41(11): 110-118.
[13] 吴函蓉,王莹,杨力,葛瑶,范玲. 我国生物技术基地平台现状与发展建议[J]. 中国生物工程杂志, 2021, 41(11): 119-123.
[14] 刘天义,冯卉,SALSABEELYousuf,解领丽,苗向阳. lncRNA在动物脂肪沉积中的研究进展*[J]. 中国生物工程杂志, 2021, 41(11): 82-88.
[15] 薛志勇,代红生,张显元,孙艳颖,黄志伟. 表达透明颤菌血红蛋白基因对酿酒酵母生长及细胞内氧化状态的影响*[J]. 中国生物工程杂志, 2021, 41(11): 32-39.