表面暴露幽门螺杆菌蛋白的生物素化和纯化

Bradley J. Voss; Timothy L. Cover

doi:10.21769/BioProtoc.1455

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Peer-reviewed

Biotinylation and Purification of Surface-exposed Helicobacter pylori Proteins

表面暴露幽门螺杆菌蛋白的生物素化和纯化

BV Bradley J. Voss

TC Timothy L. Cover email

发布: 2015年04月20日第5卷第8期 DOI: 10.21769/BioProtoc.1455 浏览次数: 7997

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Journal of Bacteriology, featuring study using the protocol.

Jul 2014

实验方案合集

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Abstract

Interactions between pathogenic bacteria and host cells are often mediated by proteins found on the surfaces of the bacteria. The Gram-negative bacterium Helicobacter pylori is predicted to produce at least 50 surface-exposed outer membrane proteins, but there has been relatively little progress in experimentally analyzing the cell-surface proteome of this organism. Herein, we describe in detail a protocol that allows biotinylation and purification of surface-exposed H. pylori proteins. A comparative analysis of surface-exposed proteins identified by this biotinylation-based approach and by several other independent methods is described in a recent publication (Voss et al., 2014).

Keywords: Outer membrane proteins (外层膜蛋白)

Autotransporter (汽车运输船)

Type V secretion (V型分泌)

Adhesin (最高)

Helicobacter pylori (幽门螺杆菌)

Materials and Reagents

Tryptone (BD, catalog number: 211705 )
Proteose peptone #3 (BD, catalog number: 211693 )
Yeast Extract (BD, catalog number: 212720 )
NaCl (Acros Organics, catalog number: 207790250 )
Dextrose (Thermo Fisher Scientific, catalog number: D14-212 )
Fetal bovine serum (Atlanta Biologicals, catalog number: S11150 )
Tris Base (Thermo Fisher Scientific, catalog number: BP152 )
Na₂HPO₄ (Sigma-Aldrich, catalog number: S5136 )
KCl (Sigma-Aldrich, catalog number: P-9333 )
10x PBS (Corning, catalog number: 46-013-CM )
MgCl₂^.6H₂O (Thermo Fisher Scientific, catalog number: M35 )
CaCl₂ (Sigma-Aldrich, catalog number: C-4901 )
EDTA-Na₂^.2H₂O (Sigma-Aldrich, catalog number: E4884 )
D-biotin (Sigma-Aldrich, catalog number: B4501 )
S-NHS-LC-Biotin (Thermo Fisher Scientific, catalog number: 21335 )
Zwittergent 3-14 (Fluka, catalog number: 40772 )
Protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693159001 )
Monomeric avidin magnetic beads, Blocking buffer, Regeneration buffer (Bioclone, catalog number: MMI-101 )
100% (w/v) TCA (Sigma-Aldrich, catalog number: T0699 )
Acetone (Thermo Fisher Scientific, catalog number: A16F )
Brucella broth (see Recipes)
Biotinylation buffer (see Recipes)
TNKCM (see Recipes)
Lysis buffer (see Recipes)
TKE (see Recipes)
TKEZ (see Recipes)
Sample dilution buffer (see Recipes)
Blocking buffer (see Recipes)
Regeneration buffer (see Recipes)
Wash buffer (see Recipes)
Elution buffer (see Recipes)

Equipment

CO₂ shaking incubator (ATR Biotech, model: AJ125B )
Bench top centrifuge (Thermo Fisher Scientific, catalog number: 75004381 )
Sonicator (Thermo Fisher Scientific, catalog number: FB505 )
Bench top centrifuge (Eppendorf, catalog number: 5424 )
Ultracentrifuge (Beckman Coulter, catalog number: A94469 )
Barnstead/Thermolyne Labquake Shaker Rotisserie (Labquake, catalog number: C400110 )
DynaMag^TM- Spin Magnet (Life Technologies, catalog number: 12320D )

Procedure

English

中文翻译

文章信息

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如何引用

Voss, B. J. and Cover, T. L. (2015). Biotinylation and Purification of Surface-exposed Helicobacter pylori Proteins. Bio-protocol 5(8): e1455. DOI: 10.21769/BioProtoc.1455.