AcrB et al.: Obstinate contaminants in a picogram scale. One more bottleneck in the membrane protein structure pipeline.
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.
Psakis
Georgios
G
Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse, 35033 Marburg, Germany. georgios.psakis@bot3.bio.unigiessen.de
Polaczek
Julia
J
Essen
Lars-Oliver
LO
D016477:Artifacts; D003460:Crystallization; D003902:Detergents / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D016480:Helicobacter pylori / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D011494:Protein Kinases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013385:Succinate Dehydrogenase / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry
D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D013431:Sulfates; D013385:Succinate Dehydrogenase; D011494:Protein Kinases; C065271:arcB protein, E coli