Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies.
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.
Wang
Da-Neng
DN
Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA. wang@saturn.med.nyu.edu
Safferling
Markus
M
Lemieux
M Joanne
MJ
Griffith
Heather
H
Chen
Yong
Y
Li
Xiao-Dan
XD
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003470:Culture Media; D003902:Detergents; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D005822:Genetic Vectors; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011786:Quality Control; D013696:Temperature
D001426:Bacterial Proteins; D003470:Culture Media; D003902:Detergents; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins