Assays for membrane proteins

Abstract

A method for screening conditions for producing and purifying transmembrane proteins using cell-free protein synthesis. The transmembrane proteins, or integrated membrane proteins (IMPs) are fused with a binding tag and expressed in a variety of potential solubilising agents before affinity purification to quantify the produced protein. The solubilising agents may be nanodiscs including fluorescently labelled nanodiscs or detergents including tandem facial amphiphile detergents. During the assay the expressed protein may be purified using affinity binding through a His-tag, the labelled nanodiscs maybe be used to quantify the amount of purified protein or the proteins may be expressed with a detector tag wherein a detector moiety is added to the composition to quantify the expressed soluble protein. There is also a kit for cell free expression of proteins comprising the nucleic acid constructs for expression of a protein fused to an affinity tag, a digital microfluidic device, nanodiscs or detergents acting as solubilising agents and solid supports for affinity purification. [No Figure]