Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies

Abstract

Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years.

Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein

“…During the last decade increasing efforts to express prokaryotic and eukaryotic membrane proteins have resulted in significant advancements for the expression of several bacterial transporter proteins [3][4][5][6][7][8][9], outer membrane proteins of bacteria [10], membrane protein complexes [11][12][13] and a few eukaryotic G-protein coupled receptors [14][15][16][17][18][19][20][21]. With others we have successfully expressed more than 70 membrane proteins from Mycobacterium tuberculosis in Escherichia coli using a His-tag [22].…”

Quality control of overexpressed membrane proteins

“…The amount of well folded protein produced in the membrane can often be increased by systematic optimization of several parameters such as the expression strain, induction temperature, growth medium, and promoter strength (4). Evaluation of conditions may be done by determining the amount of protein present in isolated membrane vesicles or by monitoring the activity of the protein, provided suitable activity assays are available.…”

Investigating transport proteins by solid state NMR

“…Overexpression of bacterial transporters has been repeatedly demonstrated (Auer et al 2001;Curnow et al 2004;Masi et al 2003;Xie et al 2004;Yerushalmi et al 1995). A more detailed summary of expression levels of various secondary membrane transporters is given in a review by Wang et al (2003).…”

“…Protein homogeneity can also be evaluated using analytical size exclusion chromatography and mass spectrometry. A very good review surveying sample preparation and optimization was written by Wang et al (2003).…”

“…Escherichia coli, yeast and baculovirus) optimised for membrane proteins, the availability of detergent screens and advanced purification technologies, in addition to the sequencing of a number of prokaryotic and eukaryotic genomes. For a review, see (Wang et al 2003). An overview of sample preparation and biophysical investigations possible at each step is given in Fig.…”