A systematic assessment of mature MBP in membrane protein production: Overexpression, membrane targeting and purification

► Efficient expression of small helical membrane proteins with a mature MBP fusion in E. coli membranes. ► Positively charged tags (not necessarily His tags) on the N-terminus enhances expression of small helical membrane proteins. ► The mMBP fusion works best for helical membrane proteins having ⩽3 transmembrane helices and <20 kDa molecular weight. ► Frequently, one-step purification can be achieved using Ni2+-NTA chromatography following cleavage of the fusion protein.