Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.
Bibliographical note© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
Funding: Biotechnology and Biological Sciences Research Council (BBSRC; via grants BB/N007417/1, BB/P025927/1 and BB/P022685/1 to RMB) and the Innovative Medicines Joint Undertaking under Grant Agreement number 115583 to the ND4BB ENABLE Consortium to RMB. EC Membrane Protein Consortium (E-MeP, FP6 LSHG-CT-2004-504601), the European Drug Initiative for Channels and Transporters (EDICT, FP7 Health-F4-2007-201924 and BBBSRC grant BB/D524832/1 to RMB and PJFH. MVD’s PhD studentship was funded by BBSRC iCASE BB/H016244/1 with Glycoform Ltd. PJFH is grateful to the Leverhulme Trust for an Emeritus Research Fellowship (Grant number EM-2014-045). BBSRC (MPSI BBS/B/14418), the Wellcome Trust (JIF 062164/Z/00/Z) and the University of Leeds. ‘Initiative d'Excellence’ program from the French State (Grant ‘DYNAMO’, ANR-11-LABEX-0011-01). PhD fellowship funded by the French Ministry of Research and Education.
- Recombinant membrane proteins
- Expression plasmid vector