Filling the Gap: A streamlined approach for monitoring expression and purification of membrane proteins with a periplasmic C‐terminus via GFP fluorescence

Abstract

Crystal structures of membrane proteins are notoriously difficult to obtain. These proteins reside in phospholipid bilayers making them difficult to express, purify, stabilize and structurally resolve. Previously, inner membrane proteins (IMP) have been recombinantly expressed and monitored using a C‐terminal green fluorescent protein (GFP) fusion in Escherichia coli. This method proved extremely useful, but is only effective for IMP with C‐termini localizing to the cytoplasm (CI), where the GFP folds correctly. To fill the gap for IMP with C‐termini localizing to the periplasm (CO), we developed the expression vectors: pWarf(‐) and pWarf(+). While pWarf(‐) fuses GFP to the C‐terminus of the IMP, pWarf(+) fuses the single transmembrane helix protein Glycophorin A and subsequent GFP to the IMP. The pWarf(+) vector effectively converts CO IMP to CI IMP. Eleven E. coli IMP from three families were selected for their predicted CO topology. High level whole cell fluorescence was observed only in the pWarf(+) system. Using the pWarf(+) vector, overexpression was rapidly screened, detergents were selected by fluorescent size exclusion chromatography and stability was monitored. Large scale purification is in progress for crystallization trials. By developing the pWarf vector system, a comprehensive, streamlined approach to monitoring overexpression, purification and stabilization has been achieved for IMP. Funding from AHA.