The funnel approach to the precrystallization production of membrane proteins.

ABSTRACT: 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 succes

SUBMITTER: Lewinson O 

PROVIDER: S-EPMC2362151 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature