Combinatorial method for overexpression of membrane proteins in Escherichia coli.
Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.
Leviatan
Shani
S
Department of Biochemistry, the George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Sawada
Keisuke
K
Moriyama
Yoshinori
Y
Nelson
Nathan
N
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005821:Genetic Techniques; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D017434:Protein Structure, Tertiary; D051381:Rats
D001426:Bacterial Proteins; D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins