利用生物素连接酶和单价链霉亲和素对活的哺乳动物细胞中的蛋白质进行成像。
Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.
作者信息
Howarth Mark, Ting Alice Y
机构信息
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
出版信息
Nat Protoc. 2008;3(3):534-45. doi: 10.1038/nprot.2008.20.
Abstract
This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the cross-linking of biotinylated targets disrupts many of streptavidin's applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d.
摘要
本方案描述了一种在哺乳动物细胞上用生物物理探针标记特定细胞表面蛋白的简单有效方法。用15个氨基酸的肽标记的细胞表面蛋白被大肠杆菌生物素连接酶（BirA）生物素化，而内源性蛋白则未被修饰。生物素基团随后允许链霉亲和素偶联物进行灵敏且稳定的结合。本方案描述了BirA和链霉亲和素用于位点特异性标记的最佳使用方法，以及如何制备BirA和单价链霉亲和素。链霉亲和素是四价的，生物素化靶标的交联会干扰链霉亲和素的许多应用。单价链霉亲和素只有一个功能性生物素结合位点，但保留了野生型链霉亲和素的飞摩尔亲和力、低解离速率和高热稳定性。位点特异性生物素化和链霉亲和素染色只需几分钟，而BirA的表达需要4天，单价链霉亲和素的表达需要8天。
相似文献
1
Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.利用生物素连接酶和单价链霉亲和素对活的哺乳动物细胞中的蛋白质进行成像。
Nat Protoc. 2008;3(3):534-45. doi: 10.1038/nprot.2008.20.
2
In vivo biotinylation of bacterial magnetic particles by a truncated form of Escherichia coli biotin ligase and biotin acceptor peptide.通过截短形式的大肠杆菌生物素连接酶和生物素接受肽在体内生物素化细菌磁性颗粒。
Appl Environ Microbiol. 2010 Sep;76(17):5785-90. doi: 10.1128/AEM.00916-10. Epub 2010 Jul 9.
3
In vivo biotinylation of the major histocompatibility complex (MHC) class II/peptide complex by coexpression of BirA enzyme for the generation of MHC class II/tetramers.通过共表达BirA酶对主要组织相容性复合体（MHC）II类/肽复合物进行体内生物素化，以生成MHC II类四聚体。
Hum Immunol. 2004 Jul;65(7):692-9. doi: 10.1016/j.humimm.2004.04.001.
4
Site-specific biotinylation of purified proteins using BirA.使用BirA对纯化蛋白质进行位点特异性生物素化。
Methods Mol Biol. 2015;1266:171-84. doi: 10.1007/978-1-4939-2272-7_12.
5
A one-process production of completely biotinylated proteins in a T7 expression system.在 T7 表达系统中一步法生产完全生物素化的蛋白质。
Biotechnol Appl Biochem. 2024 Oct;71(5):1070-1078. doi: 10.1002/bab.2598. Epub 2024 May 21.
6
Displaying non-natural, functional molecules on yeast surfaces via biotin-streptavidin interaction.通过生物素-链霉亲和素相互作用在酵母表面展示非天然功能分子。
J Biotechnol. 2010 Jan 1;145(1):79-83. doi: 10.1016/j.jbiotec.2009.10.011.
7
Noncovalent immobilization of streptavidin on in vitro- and in vivo-biotinylated bacterial magnetic particles.链霉亲和素在体外和体内生物素化细菌磁性颗粒上的非共价固定。
Appl Environ Microbiol. 2008 Aug;74(16):5139-45. doi: 10.1128/AEM.00618-08. Epub 2008 Jun 20.
8
Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation.用于体外生物素化的大肠杆菌BirA生物素连接酶的表达与纯化
Protein Expr Purif. 2012 Mar;82(1):162-7. doi: 10.1016/j.pep.2011.12.008. Epub 2012 Jan 2.
9
The wing of a winged helix-turn-helix transcription factor organizes the active site of BirA, a bifunctional repressor/ligase.一个具有翼状螺旋-转角-螺旋结构的转录因子的翅膀组织了双功能抑制剂/连接酶 BirA 的活性位点。
J Biol Chem. 2013 Dec 13;288(50):36029-39. doi: 10.1074/jbc.M113.525618. Epub 2013 Nov 4.
10
Biotinylation of Membrane Proteins for Binder Selections.用于结合物选择的膜蛋白生物素化。
Methods Mol Biol. 2020;2127:151-165. doi: 10.1007/978-1-0716-0373-4_11.
引用本文的文献
1
Merbecovirus S2 subunit vaccines elicit cross reactive antibodies and provide partial protection against MERS coronavirus.默贝科病毒S2亚基疫苗可引发交叉反应性抗体，并对中东呼吸综合征冠状病毒提供部分保护。
Npj Viruses. 2025 Jul 29;3(1):60. doi: 10.1038/s44298-025-00142-9.
2
Fluorescent labeling of proteins in vitro and in vivo using encoded peptide tags.使用编码肽标签在体外和体内对蛋白质进行荧光标记。
J Biol Chem. 2025 May 14;301(6):110229. doi: 10.1016/j.jbc.2025.110229.
3
Identification of E3 Ubiquitin Ligase Substrates Using Biotin Ligase-Based Proximity Labeling Approaches.使用基于生物素连接酶的邻近标记方法鉴定E3泛素连接酶底物
Biomedicines. 2025 Apr 2;13(4):854. doi: 10.3390/biomedicines13040854.
4
Fluorescent labeling strategies for molecular bioimaging.用于分子生物成像的荧光标记策略。
Biophys Rep (N Y). 2025 Mar 12;5(1):100200. doi: 10.1016/j.bpr.2025.100200. Epub 2025 Feb 12.
5
Multivalent S2 subunit vaccines provide broad protection against Clade 1 sarbecoviruses in female mice.多价S2亚基疫苗为雌性小鼠提供针对第1进化枝严重急性呼吸综合征冠状病毒的广泛保护。
Nat Commun. 2025 Jan 7;16(1):462. doi: 10.1038/s41467-025-55824-y.
6
Single immunization with an influenza hemagglutinin nanoparticle-based vaccine elicits durable protective immunity.用基于流感血凝素纳米颗粒的疫苗进行单次免疫可引发持久的保护性免疫。
Bioeng Transl Med. 2024 Jun 3;9(5):e10689. doi: 10.1002/btm2.10689. eCollection 2024 Sep.
7
Ebola Virus Glycoprotein Strongly Binds to Membranes in the Absence of Receptor Engagement.埃博拉病毒糖蛋白在不与受体结合的情况下强烈结合到膜上。
ACS Infect Dis. 2024 May 10;10(5):1590-1601. doi: 10.1021/acsinfecdis.3c00622. Epub 2024 Apr 29.
8
Understanding the nervous system: lessons from Frontiers in Neurophotonics.理解神经系统：来自《神经光子学前沿》的经验教训。
Neurophotonics. 2024 Jan;11(1):014415. doi: 10.1117/1.NPh.11.1.014415. Epub 2024 Mar 27.
9
Broad protection against clade 1 sarbecoviruses after a single immunization with cocktail spike-protein-nanoparticle vaccine.单次免疫鸡尾酒型刺突蛋白纳米颗粒疫苗可提供针对 1 型沙贝科病毒属的广泛保护。
Nat Commun. 2024 Feb 12;15(1):1284. doi: 10.1038/s41467-024-45495-6.
10
Infrared photoinduced electrochemiluminescence microscopy of single cells.单细胞的红外光致电化学发光显微镜术
Chem Sci. 2023 Dec 8;15(6):2055-2061. doi: 10.1039/d3sc05983a. eCollection 2024 Feb 7.
本文引用的文献
1
Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.利用小分子探针重定向硫辛酸连接酶用于细胞表面蛋白标记
Nat Biotechnol. 2007 Dec;25(12):1483-7. doi: 10.1038/nbt1355. Epub 2007 Dec 2.
2
Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems: toward functional protein arrays.使用体外、体内和无细胞系统进行位点特异性蛋白质生物素化的策略：迈向功能性蛋白质阵列
Nat Protoc. 2006;1(5):2386-98. doi: 10.1038/nprot.2006.338.
3
Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum.通过保留在内质网中的生物素连接酶对重组抗体进行代谢生物素化。
Biomol Eng. 2007 Sep;24(3):283-91. doi: 10.1016/j.bioeng.2007.02.003. Epub 2007 Feb 15.
4
Purine and pyrimidine receptors.嘌呤和嘧啶受体。
Cell Mol Life Sci. 2007 Jun;64(12):1471-83. doi: 10.1007/s00018-007-6497-0.
5
Imaging intracellular fluorescent proteins at nanometer resolution.以纳米分辨率成像细胞内荧光蛋白。
Science. 2006 Sep 15;313(5793):1642-5. doi: 10.1126/science.1127344. Epub 2006 Aug 10.
6
Chemical labeling strategies for cell biology.细胞生物学的化学标记策略。
Nat Methods. 2006 Aug;3(8):591-6. doi: 10.1038/nmeth906.
7
Membrane lateral diffusion and capture of CFTR within transient confinement zones.膜侧向扩散以及囊性纤维化跨膜传导调节因子在瞬时受限区域内的捕获
Biophys J. 2006 Aug 1;91(3):1046-58. doi: 10.1529/biophysj.106.084830. Epub 2006 May 19.
8
Metabolic biotinylation of cell surface receptors for in vivo imaging.用于体内成像的细胞表面受体的代谢生物素化
Nat Methods. 2006 May;3(5):391-6. doi: 10.1038/nmeth875.
9
A monovalent streptavidin with a single femtomolar biotin binding site.一种具有单个飞摩尔生物素结合位点的单价抗生物素蛋白。
Nat Methods. 2006 Apr;3(4):267-73. doi: 10.1038/nmeth861.
10
High-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes.使用生物素标记噬菌体和量子点纳米复合物进行高灵敏度细菌检测。
Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):4841-5. doi: 10.1073/pnas.0601211103. Epub 2006 Mar 20.
Zotero 插件