Use of Escherichia coli for the Production and Purification of Membrane Proteins
Individual types of ion channels and other membrane proteins are typically expressed only at low levels in their native membranes, rendering their isolation by conventional purification techniques difficult. The heterologous over-expression of such protei
- PDF / 430,538 Bytes
- 22 Pages / 504.57 x 720 pts Page_size
- 22 Downloads / 213 Views
1
Introduction Twenty to thirty percent of genes in most living organisms encode membrane proteins (1), which play critical roles in many aspects of biology, ranging from cell signalling to nutrition. Moreover, such proteins represent the targets of more than 50% of currently used therapeutic drugs (2). Isolation of membrane proteins for structural and functional analysis is therefore of great biological and medical importance. However, individual types of ion channels, transporters, and other membrane proteins typically represent less than 1% of the total protein content of natural biological membranes and this low level of expression hinders their purification. Moreover, the nonionic detergents required to solubilize the proteins can interfere with purification by conventional approaches, such as ion-exchange chromatography, by shielding some of the charged groups on the protein surface within the hydrophilic head
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_3, © Springer Science+Business Media, LLC 2013
33
34
Vincent G.L. Postis et al.
groups of the detergents in the protein-detergent micelle. Typically, isolation of membrane proteins in amounts sufficient for structural and other investigations therefore requires their over-expression, bearing an affinity tag, in a suitable heterologous expression system. Choice of the type of affinity tag and the location of its attachment must be carefully considered, because these can influence the synthesis and insertion of the protein in the membrane (3). Choice of expression system is also important. For example, while some human membrane proteins can be functionally expressed in bacteria (4), high-level expression usually requires a eukaryotic host (5). Additionally, the choice of detergent used for protein solubilization is often critical in order that the protein not only remains in a soluble, non-aggregated state for prolonged periods of time but also that it retains its native structure and function. Here we describe methods that can be employed for the expression of membrane proteins in Escherichia coli and for their subsequent purification. Whilst most applicable to prokaryote membrane proteins, including homologues of human ion channels and transporters, the procedures involved can be adapted for the purification of proteins expressed in eukaryotic systems such as mammalian and insect cells or yeasts.
2
Materials
2.1 Reagents and Buffers for Bacterial Cell Culture and Membrane Preparation
1. E. coli host strains: BL21-gold(DE3) (Stratagene), BL21 Star™ (DE3) (Invitrogen), and C43(DE3) (Lucigen Corporation), all harboring the plasmid pRARE2 (Novagen) if required (see Note 1). 2. Expression constructs: These should contain the open reading frame (ORF) for the protein of interest, bearing appropriate N- and/or C-terminal affinity tags, under the control of a lactoseinducible tac promoter (i.e., in a derivative of the vector pTTQ18 (6)) or another suitable promoter (see Note
Data Loading...
