An Improved Strategy for Fluorescent Tagging of Membrane Proteins for Overexpression and Purification in Mammalian Cells.

Abstract

An essential prerequisite for in vitro biochemical or structural studies is a construct that is amenable to high level expression and purification and is biochemically "well-behaved". In the field of membrane protein research, the use of green fluorescent protein (GFP) to monitor and optimize the heterologous expression in different hosts has radically changed the ease of streamlining and multiplexing the testing of a large number of candidate constructs. This is achieved by genetically fusing the fluorescent proteins to the N- or C-terminus of the proteins of interest to act as reporters which can then be followed by methods such as microscopy, spectroscopy, or in-gel fluorescence. Nonetheless, a systematic study on the effect of GFP and its spectral variants on the expression and yields of recombinant membrane proteins is lacking. In this study, we genetically appended four common fluorescent protein tags, namely, mEGFP, mVenus, mCerulean, and mCherry, to the N- or C-terminus of different membrane proteins and assessed their expression in mammalian cells by fluorescence-detection size exclusion chromatography (FSEC) and protein purification. We find that, of the four fluorescent proteins, tagging with mVenus systematically results in higher expression levels that translates to higher yields in preparative purifications, thus making a case for switching to this yellow spectral variant as a better fusion tag.