Related Experiment Video
Updated: Jun 26, 2026
Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis
Published on: July 16, 2020
AcrB et al.: Obstinate contaminants in a picogram scale. One more bottleneck in the membrane protein structure
Georgios Psakis1, Julia Polaczek, Lars-Oliver Essen
1Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse, 35033 Marburg, Germany. georgios.psakis@bot3.bio.unigiessen.de
Abstract:
Heterologous expression of integral membrane proteins from Helicobacter pylori 26695 in Escherichia coli enabled the identification of 17 candidates for purification and subsequent crystallization. 45% of the purified proteins were contaminated with what was later identified as the multidrug efflux pump (AcrB)of E. coli, and 17% with the succinate dehydrogenase. While additional purification steps ensured removal of succinate dehydrogenase, they failed to remove AcrB completely, leaving picogram amounts present infractions intended for 3D-crystallization. Two of these targets, the Na+ dependent D-glucose/D-galactose transporter (GluP-HP1174) and the carbon starvation protein A (CstA-HP1168), produced small crystals(<40 lm). Crystals from the GluP preparation diffracted to 4.2 A resolution and belonged to the rhombohedral space group H32. Subsequent molecular replacement proved that these crystals were derived from a contaminant, the efflux transporter AcrB. This unexpected crystallization of AcrB from picogram amounts was observed in six new conditions. The systematic occurrence of AcrB in membrane preparations stems from the upregulation of its transcription in response to the stress induced by the expression of a selected target. This, along with its tendency to crystallize in the picogram scale, poses a serious concern in membrane protein expression using heterologous hosts harbouring AcrB.
Insights
Contaminant proteins, like E. coli's AcrB efflux pump, can crystallize unexpectedly during Helicobacter pylori membrane protein purification. Picogram amounts of AcrB hinder structural studies by mimicking target proteins.
Area of Science:
- Structural Biology
- Membrane Protein Biochemistry
- Microbiology
Background:
- Heterologous expression is crucial for studying integral membrane proteins.
- Helicobacter pylori proteins are targets for understanding bacterial physiology.
- Escherichia coli is a common host for heterologous protein expression.
Purpose of the Study:
- To identify and purify integral membrane proteins from Helicobacter pylori 26695.
- To investigate challenges in crystallizing these proteins using Escherichia coli as a host.
- To characterize protein contaminants affecting structural studies.
Main Methods:
- Heterologous expression of H. pylori proteins in E. coli.
- Protein purification techniques including chromatography.
- Crystallization trials and X-ray diffraction analysis.
- Molecular replacement for structure determination.
Main Results:
- 17 H. pylori integral membrane protein candidates were identified.
- Contamination by E. coli's AcrB efflux pump and succinate dehydrogenase was observed.
- AcrB contamination persisted despite purification, hindering crystallization of target proteins.
- Unexpected crystallization of AcrB from picogram quantities was achieved in multiple conditions.
Conclusions:
- The multidrug efflux pump AcrB from E. coli can contaminate H. pylori membrane protein preparations.
- AcrB's tendency to crystallize from trace amounts poses a significant challenge for structural studies.
- Upregulation of AcrB transcription under expression stress contributes to its prevalence.
- Careful validation is needed to distinguish contaminant crystals from target protein crystals.
Related Concept Videos
01:09Multi-pass Transmembrane Proteins and β-barrels
α-Helix containing multi-pass transmembrane proteins
Multi-pass transmembrane proteins such as G-protein-linked receptors (GPCRs) and...
01:18Detergent Purification of Membrane Proteins