Item

Abstract

Cell-free protein production offers a versatile alternative to complement in vivo expression systems. However, usage of prokaryotic cell-free systems often leads to non-functional proteins. We modified a previously designed cell-free system based on the protozoan Leishmania tarentolae, a parasite of the Moorish gecko Tarentola mauritanica, together with a species-independent translational sequences-based plasmid to produce human membrane proteins in 2 hours reaction time. We successfully established all four commonly used expression modes for cell-free synthesis of membrane proteins with a human organic anion transporter, SLC17A3, as a model membrane protein: (i) As precipitates without the addition of any hydrophobic environment, (ii) in the presence of detergents, (iii) with the addition of liposomes, and (iv) supplemented with nanodiscs. We utilized this adapted system to synthesize 22 human solute carriers from 20 different families. Our results demonstrate the capability of the Leishmania tarentolae cell-free system for the production of a huge variety of human solute carriers in the precipitate mode. Furthermore, purified SLC17A3 shows the formation of an oligomeric state.