Cell-free Protein Expression of Membrane Proteins Using Nanolipoprotein Particles

Abstract

The paucity of membrane protein science reflects the hurdles that these proteins impose on their expression and purification. Expression of membrane proteins in vivo frequently results in cell toxicity, protein aggregation, misfolding, and low yield. Vesicle preparation, detergent extraction, and membrane protein reconstitution into liposomes are processes that inexorably lead to constraints on protein accessibility, surface immobilization, and activity assays. We report a cell-free protein expression approach for expressing and inserting integral membrane proteins into water soluble particles (brand name MembraneMaxTM, offered tag-free or histidine tagged) composed of a phospholipid membrane bilayer surrounded by a scaffold protein. The method circumvents the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer.