Effective high-throughput overproduction of membrane proteins in Escherichia coli
Artikel i vetenskaplig tidskrift, 2008

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-β-d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2 mg/l, with 18 targets producing at levels of 5 mg/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.

Membrane proteins

Legionnaires’ disease

Legionella pneumophila

Escherichia coli

His-tag

Structural genomics

Auto-induction

Protein production

Författare

Euan Gordon

Chalmers, Kemi- och bioteknik, Molekylär mikroskopi

Rob Horsefield

Göteborgs universitet

Herman G.P. Swarts

Nijmegen Centre for Molecular Life Sciences - NCMLS

Jan Joep H.H.M. de Pont

Nijmegen Centre for Molecular Life Sciences - NCMLS

Richard Neutze

Göteborgs universitet

Arjan Snijder

Chalmers, Kemi- och bioteknik, Molekylär mikroskopi

Protein Expression and Purification

1046-5928 (ISSN) 1096-0279 (eISSN)

Vol. 62 1 1-8

Ämneskategorier

Kemi

DOI

10.1016/j.pep.2008.07.005

PubMed

18692139