Membrane proteins have crucial roles in many cellular and physiological processes. Understanding the structures and functions of these proteins is academically and commercially important. However, the lack of efficient methods for expressing membrane proteins has created a bottleneck. While one third of all proteins are membrane proteins, less than 100 membrane protein structures have been solved to the date. Among membrane proteins, G protein coupled receptors (GPCR) represent a large family of about 1000 proteins in human cells and are the most important drug targets. We propose the development of the SUMO-Fusion system to enhance expression and purification of a wide variety of membrane proteins in E. coli. The key features of the SUMO Fusion technology are: 1) attachment of the C-terminus of SUMO protein to the N terminus of a membrane protein to act as a chaperone, enhancing the qualitative and quantitative expression of the membrane protein; 2) introduction of N-terminal 6xHis-SUMO to act as a tag to allow rapid purification; 3) exploitation of SUMO proteases to provide efficient cleavage of SUMO fusions, thereby generating membrane proteins with native N-termini. The goal of this proposal is to develop and improve the SUMO fusion system for enhancement of expression and purification of model membrane proteins in E. coli.
