Expression, Solubilization, and Purification of Bacterial Membrane Proteins

Constance J. Jeffery1

1 Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.15
DOI:  10.1002/0471140864.ps2915s83
Online Posting Date:  February, 2016
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Abstract

Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein‐detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification. © 2016 by John Wiley & Sons, Inc.

Keywords: membrane protein; alpha‐helical; transmembrane protein; integral membrane protein; purification; solubilization; detergent

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Expression, Solubilization, and Purification of Bacterial Membrane Proteins Using IMAC
  • Support Protocol 1: Further Purification of a Membrane Protein Using Gel Filtration Chromatography
  • Support Protocol 2: Identifying Detergent for Membrane Protein Solubilization
  • Reagents And Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Expression, Solubilization, and Purification of Bacterial Membrane Proteins Using IMAC

  Materials
  • Luria broth (LB) (see recipe)
  • Kanamycin stock solution (see recipe), or other appropriate antibiotic to select the plasmid encoding transmembrane protein of interest
  • Frozen stock of E. coli expression strain harboring the expression plasmid that encodes the membrane protein of interest fused to a 6His affinity tag
  • 2× YT in Fernbach flasks (see recipe)
  • 1× SDS sample buffer (see recipe)
  • 1 M IPTG (see recipe)
  • Cell resuspension buffer (see recipe)
  • Membrane resuspension buffer (see recipe)
  • DDM, n‐dodecyl‐β‐D‐maltopyranoside (Anatrace, cat. no. D310)
  • Co2+ Talon Metal Affinity Resin (Takara Clontech, cat. no. 635501)
  • IMAC binding and wash buffer (see recipe)
  • 2× SDS sample buffer (see recipe)
  • IMAC elution buffer (see recipe)
  • Bio‐Safe Coomassie Stain (BioRad, cat. no. 161‐0786)
  • BCA Protein Assay Kit (Thermo Scientific Pierce, cat. no. 23225)
  • Test tubes, 10‐ml to 15‐ml (autoclaved)
  • Roller at 37°C
  • 500‐ml flask (autoclaved)
  • Shakers at 37°C and 30°C
  • Spectrophotometer
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge (e.g., Eppendorf, cat. no. 5424R)
  • Large‐scale centrifuge that holds 1 liter centrifuge bottles (e.g., Sorvall RC3B Plus with H6000A rotor)
  • 1‐liter centrifuge bottles
  • French pressure cell (GlenMills, cat. no. 6800‐FA‐032)
  • French press (e.g., GlenMills)
  • Bottles, 100 ml
  • Medium speed centrifuge (e.g., Sorvall™ RC 6 Plus)
  • SS34 rotor
  • SS34 centrifuge tubes with appropriate caps (Thermo Scientific, cat. no. 3119‐0030)
  • T‐647.5 ultracentrifuge rotor
  • Ultracentrifuge (e.g., Sorvall WX 80+)
  • T‐647.5 ultracentrifuge tubes with caps (Seton Scientific, cat. no. 5097)
  • High vacuum grease
  • Rocker at 4°C
  • Column body, 1 × 10‐cm (BioRad, cat. no. 7374011)
  • 10‐ml pipets (Sigma‐Aldrich, cat. no. CLS4492‐200EA)
  • Tongue depressors
  • Additional reagents and equipment for SDS gel electrophoresis of proteins (unit 10.1; Gallagher, ), including 10% polyacrylamide SDS‐PAGE gels, SDS‐PAGE running buffer, protein molecular weight markers (BioRad, cat. no. 161‐0375)

Support Protocol 1: Further Purification of a Membrane Protein Using Gel Filtration Chromatography

  Additional Materials (also see Basic Protocol 1)
  • Pooled fractions of protein‐detergent complex solution from Basic Protocol 1, step 24
  • Filtered dH 2O
  • Gel filtration running buffer (see recipe)
  • Filtered 20% (v/v) ethanol (EtOH)
  • Spin concentrator, for example Millipore Amicon Ultra‐15 (Millipore, cat. no. UFC903008)
  • Akta chromatography system with fraction collector
  • 240‐μl loading loop for Akta system
  • Superose 12 30/10 column (GE Healthcare, cat. no. 17‐5173‐01)
  • Syringe (Fisher, cat. no. 14‐823‐16D)
  • Syringe filter (Sigma‐Aldrich, cat. no. Z359904‐50EA)
  • Test tubes that fit in fraction collector (Fisher, cat. no. 14‐959‐70C)

Support Protocol 2: Identifying Detergent for Membrane Protein Solubilization

  Additional Materials (also see Basic Protocol 1)
  • Membrane suspension from Basic Protocol 1, step 15
  • Detergent stock solutions (see reciperecipes)
  • TLA100.3 rotor
  • Ultracentrifuge tubes for TLA100.3 rotor ((Beckman Coulter, cat. no. 349622)
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.jove.com/video/2134/expression‐deterxgent‐solubilization‐purification‐membrane‐transporter
  This video illustrates the purification of a recombinant bacterial integral membrane protein, the MexB protein from Pseudomonas aeruginosa, expressed in E. coli (Bhatt and Jeffery, ).
  https://www.anatrace.com/Technical‐Documentation/Catalogs/Anatrace‐Detergents‐Booklet‐FINAL.aspx
  This is a link to a resource entitled Detergents and their Uses in Membrane Protein Science provided by Anatrace, Inc., Maumee, OH 43537.
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