The funnel approach to the precrystallization production of membrane proteins.
Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a "funnel approach" to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies.
Lewinson
Oded
O
Division of Chemistry and Chemical Engineering 114-96, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA.
Lee
Allen T
AT
Rees
Douglas C
DC
D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008670:Metals / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008967:Molecular Biology / Q000379:methods*; D021381:Protein Transport; D013816:Thermodynamics
D003902:Detergents; D008565:Membrane Proteins; D008670:Metals