监视分泌细菌毒力因子的组装使用站点特定的交联

Immunology and Infection

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Pavlova, O., Ieva, R., Bernstein, H. D. Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking. J. Vis. Exp. (82), e51217, doi:10.3791/51217 (2013).

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Abstract

本文介绍的方法到感兴趣的蛋白质和其他因素之间的检测和分析动态相互作用的体内 。我们的方法是基于最初是由彼得·舒尔茨和他的同事开发了1琥珀抑制技术。琥珀突变首先引入在编码目的蛋白质的基因的一个特定的密码子。琥珀突变体,然后在大肠杆菌中表达大肠杆菌连同编码基因的琥珀抑制tRNA和从詹氏甲烷球菌来源的氨基酰-tRNA合成酶。使用这种系统中,光活化的氨基酸类似物对 - 苯甲酰基苯(BPA)被结合在琥珀密码子。细胞,然后用紫外光照射到BPA残余物共价连接到蛋白质,都设在3-8埃。光交联是在用脉冲追踪标记和感兴趣的蛋白质的免疫沉淀结合,以便监测变化进行这发生在几秒钟的时间尺度来分钟蛋白质 - 蛋白质相互作用。我们优化了程序,研究了由两个独立的结构域,其被集成到外膜,并且被转运到细胞外空间中的结构域的结构域的细菌毒力因子的组件,但该方法可用于研究许多不同装配过程和生物学途径在原核和真核细胞。原则上相互作用的因素,甚至特定的相互作用结合到感兴趣的蛋白质因子的残基可以通过质谱法来识别。

Materials

Name Company Catalog Number Comments
QuikChange II Site-Directed Mutagenesis Kit Agilent 200521
BPA (H-p-Bz-Phe-OH) Bachem F-2800
TRAN35S-LABEL, Metabolic Labeling Reagent (35S-L-methionine and 35S-L-cysteine, >1,000 Ci/mmol) MP Biomedicals 51006
Spectroline SB-100P Super-High-Intensity UV Lamp, 365 nm Spectronics Corporation SB-110P
Spectroline Replacement Bulb 100S Spectronics Corporation 11-992-15
Falcon Tissue Culture Plate, 6-well Becton Dickinson Labware 353046
Disposable 125 ml Erlenmeyer flask Corning 430421
Innova 3100 shaking water bath New Brunswick Scientific n.a.

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