Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells

Abstract

A major obstacle for studying membrane proteins by biophysical techniques is the difficulty to produce sufficient amount of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially a eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacteria and insect cells, solubilization screening, large-scale production as well as commonly used affinity chromatography purification methods. We discuss general optimisation conditions such as promoters, tags and describe current techniques that can be used in any laboratory without specialised expensive equipment. Especially for insect cells, GFP-fusions are particularly useful for localisation and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.