title: Effective high-throughput overproduction of membrane proteins in Escherichia coli.
abstract: Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli. Protein production was performed in 0.5 ml cultures in standard 24-well plates, allowing increased throughput with minimal effort. The effect of the location of a histidine purification tag was analyzed, and the effect of decreasing the length of the N- and C-terminal extensions introduced by the Gateway cloning strategy is presented. We observed that the location and length of the purification tag significantly affected protein production levels. In addition, an auto-induction protocol for membrane protein expression was designed to enhance the overproduction efficiency such that, regardless of the construct used, much higher expression was achieved when compared with standard induction approaches such as isopropyl-beta-d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2mg/l, with 18 targets producing at levels of 5mg/l or higher. In summary, we have designed a fast and efficient process for the production of medically relevant membrane proteins with a minimum number of screening parameters.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Box 462, 40530 Gothenburg, Sweden.
authors.forename: Euan
authors.initials: E
authors.lastname: Gordon
authors.forename: Rob
authors.initials: R
authors.lastname: Horsefield
authors.forename: Herman G P
authors.initials: HG
authors.lastname: Swarts
authors.forename: Jan Joep H H M
authors.initials: JJ
authors.lastname: de Pont
authors.forename: Richard
authors.initials: R
authors.lastname: Neutze
authors.forename: Arjan
authors.initials: A
authors.lastname: Snijder
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000235:genetics / Q000378:metabolism; D016952:Legionella pneumophila / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010766:Phosphorylation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine