Methods of Identifying Membrane Proteins in Spirochetes

James A. Carroll1

1 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12C.2
DOI:  10.1002/9780471729259.mc12c02s16
Online Posting Date:  February, 2010
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Abstract

Bacterial membrane proteins serve vital functions such as nutrient acquisition, sensation of the environment, and in gene regulation, secretion, and attachment. Proteins on the cell surface are instrumental in host‐pathogen interactions and many serve as immunogens that confer protection as targets for neutralizing antibodies. Integral membrane and lipidated proteins possess hydrophobic domains or lipid anchors that interact with the lipid bilayer of cellular membranes, allowing the investigator to use hydrophobicity as a means for enrichment. The spirochete Borrelia burgdorferi is a Gram negative‐like microorganism that produces many integral and lipidated proteins, several of which have proven important during infection and transmission of the bacterium from the tick vector to the mammalian host. Protocols described in this unit for enriching membrane proteins have been extensively used by investigators in the study of B. burgdorferi, but can be easily adapted to identify and characterize membrane‐associated and surface‐exposed proteins associated with other bacteria. Curr. Protoc. Microbiol. 16:12C.2.1‐12C.2.13. © 2010 by John Wiley & Sons, Inc.

Keywords: membrane protein; fractionation; Triton X‐114; surface‐exposed; protease treatment

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Sub‐Cellular Protein Fractions: Cell Lysate, Soluble, and Membrane‐Associated Proteins
  • Basic Protocol 2: Triton X‐114 Phase Partitioning for Enrichment of Lipidated and Integral Membrane Proteins
  • Alternate Protocol 1: Phase Partitioning of Cell Lysate/Membrane Vesicles
  • Basic Protocol 3: Protease Treatment of Intact Spirochetes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Sub‐Cellular Protein Fractions: Cell Lysate, Soluble, and Membrane‐Associated Proteins

  Materials
  • Bacteria (e.g, B. burgdorferi; see unit 12.1)
  • HN buffer (see recipe)
  • Complete protease inhibitor (e.g., Roche)
  • Centrifuge
  • 15‐ml Falcon snap‐cap tubes
  • French pressure cell (SLM‐Aminco)
  • Ultracentrifuge (Sorvall Discovery M150 SE, rotor: S140 AT)
  • Open‐topped, thick‐walled polyallomer tubes designed for ultracentrifugation
  • Small sterile metal spatula
  • Glass Teflon tissue homogenizer (Kontes Glass)
  • Additional reagents and equipment for culturing B. burgdorferi (unit 12.1), determining protein concentration (e.g., Markwell et al., ), performing SDS‐PAGE (Gallagher, ), staining of proteins in gels (Sasse and Gallagher, ), and immunoblotting (Gallagher et al., )

Basic Protocol 2: Triton X‐114 Phase Partitioning for Enrichment of Lipidated and Integral Membrane Proteins

  Materials
  • Spirochetes (unit 12.1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Triton X‐114
  • 100% acetone (ice‐cold)
  • Deionized water
  • Laemmli buffer (see Gallagher, )
  • Microcentrifuge at 4°C
  • Rotisserie Shaker (Labquake, Barnstead/Thermolyne)
  • 37°C water bath
  • Microcentrifuge at 37°C
  • Vortex
  • 10‐ml acetone‐resistant tubes
  • −20°C freezer
  • Additional reagents and equipment for culturing B. burgdorferi (unit 12.1), performing SDS‐PAGE (Gallagher, ), staining of proteins in gels (Sasse and Gallagher, ), and immunoblotting (Gallagher et al., )

Alternate Protocol 1: Phase Partitioning of Cell Lysate/Membrane Vesicles

  • Cell lysate or isolated membrane vesicles ( protocol 1)
  • Rotator

Basic Protocol 3: Protease Treatment of Intact Spirochetes

  Materials
  • Spirochetes (unit 12.1)
  • BSK medium (see unit 12.1)
  • HN buffer (see recipe) or PBS supplemented with 10 mm MgCl 2
  • Trypsin
  • Proteinase K
  • Pronase
  • Phenylmethylsulfonyl fluoride (PMSF; see recipe)
  • Pefabloc SC (see recipe)
  • EDTA
  • Protease inhibitor cocktail (e.g., Complete protease inhibitor; Roche)
  • Laemmli buffer (see Gallagher, )
  • Microscope with darkfield condenser
  • Centrifuge
  • Microcentrifuge
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for culturing B. burgdorferi (unit 12.1), performing SDS‐PAGE (Gallagher, ), staining of proteins in gels (Sasse and Gallagher, ), and immunoblotting (Gallagher et al., )
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Figures

  •   FigureFigure 12.C0.1 SDS‐PAGE and Coomassie stain of subcellular fractions obtained from B. burgdorferi. The cell lysate (CL) was produced by mechanical lysis of spirochetes by French pressure cell as described, and was subjected to separation of soluble proteins (SP) from membrane‐associated proteins (MP) by ultracentrifugation. Outer surface proteins A and C (OspA and OspC, respectively) and the major flagellar protein (FlaB) are indicated. Relative molecular masses in kiloDaltons are indicated to the left of the gel.
  •   FigureFigure 12.C0.2 SDS‐PAGE and silver stain of Triton X‐114 phase partitioning of intact B. burgdorferi. The insoluble material (IP) was pelleted prior to phase partitioning and separation of the aqueous phase (AP) from the detergent phase (DP) by centrifugation. B. burgdorferi cell lysate (CL) was included as a control. Lipidated outer surface proteins OspA, OspB, and OspC are indicated and partition to the detergent‐enriched phase as expected. Relative molecular masses in kiloDaltons are indicated to the left of the gel.
  •   FigureFigure 12.C0.3 SDS‐PAGE, silver stain, and immunoblot of intact B. burgdorferi treated with proteinase K (PK), trypsin (Tr), or pronase (Pn). Untreated cells (NT) were included as a control. Lipidated outer surface proteins OspA, OspB, and OspC are indicated by arrows on the silver‐stained gel image. Relative molecular masses in kiloDaltons are indicated to the left of the gel. Note that by silver staining the samples, it is evident that OspB and OspC are sensitive to all protease treatments, while OspA is resistant to trypsin and partially resistant to pronase. Panels below the silver stain demonstrate immunoblotting of identical samples with antibodies against BBA65 (a surface exposed lipoprotein), FlaB (the major flagellar subunit located in the periplasmic space), and OppA1 (an oligopeptide permease that is found in the inner membrane). Notice the lack of significant proteolytic degradation of FlaB and OppA1 during treatment, indicative of an intact and unperturbed outer membrane.

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Literature Cited

Literature Cited
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