Bridging the gap: a GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini.

ABSTRACT: Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C(in)) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C(out)) to C(in) ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C(out) topology resu

SUBMITTER: Hsieh JM 

PROVIDER: S-EPMC2867025 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature