Starting materials needed for the purification of integral membrane proteins for structural and functional studies

Extracted from Purifying Challenging Proteins - Principles and Methods, GE Healthcare, 2007

Starting material

Membrane proteins from natural sources

The natural source of a membrane protein can be considered as a starting material for purification. The only three-dimensional structure in molecular detail that has been reported to date for a eukaryotic G-protein coupled receptor (GPCR), bovine rhodopsin, was obtained with protein purified from bovine retina, where the protein is highly abundant. In many cases, however, low abundance of the target protein precludes the use of the natural source as starting material.

Examples of purifications from natural sources are presented later in this chapter.


Vectors used for the expression of soluble proteins are also commonly used for the production of membrane proteins. It is useful to design a number (10 to 50) of different constructs, including different homologues, to increase the chance that a particular membrane protein can be produced in an active form. In addition to the general considerations for choosing a vector (see page 100, Recombinant Protein Purification Handbook), a number of other aspects relate more specifically to choosing a vector for expressing membrane proteins.

Affinity tagging greatly facilitates expression screening based on chromatographic enrichment, as well as optimization and use of protocols for purification of membrane proteins. Polyhistidine tags are commonly used for membrane proteins, but the GSTtag and others have also been used successfully. The insertion of a protease cleavage site between the affinity tag and the target protein enables removal of the tag before further analyses.

While a hexahistidine tag (His6) is the standard option for water-soluble proteins, longer histidine tags (with 8 or 10 histidine residues) are often used for membrane proteins to increase the binding strength and thus improve yields in IMAC purification. Drawbacks with longer (> 6 histidine residues) histidine tags are that expression levels have been reported to be decreased in some cases and that a higher imidazole concentration is required for elution.

Tags should generally be placed on the C-terminal end of the protein to reduce risk of affecting the membrane insertion process based on the N-terminal signal peptide.

Fusion of the target membrane protein to a fluorescent protein tag such as GFP in combination with a histidine tag allows direct and convenient visualization of the target during expression, solubilization, and purification and can speed up the optimization of these processes.

Expression and screening

To correctly decide which conditions and constructs will be best suited for producing the protein for the intended study, an efficient screening protocol is essential. Because of the relatively low concentrations of overexpressed membrane proteins, it is useful to apply affinity tags combined with separation methods that allow enrichment of the target protein.

Overexpression is a major bottleneck in the overall workflow for membrane protein production. Several host systems are available and the final choice will depend both on protein-specific requirements (e.g., for post-translational modifications) and practical aspects (e.g., available equipment in the lab and expertise). It is often useful to try different hosts or host strains in parallel for a particular target protein to increase the likelihood of success. In addition, homologous membrane proteins from several sources can be cloned in parallel to be able to select those that express well.

E. coli strain BL21 (DE3) is the most commonly used host for overexpression of membrane proteins, in combination with a pET vector. “High” expression levels for functional membrane proteins are usually more than an order of magnitude lower than for overexpression of water-soluble proteins in E. coli. One inherent issue is that membrane proteins need to be inserted into membranes, and the availability of membrane structures in most cells is limited.

The issue with limited membrane availability can be addressed by using a host with large amounts of internal membranes (e.g., Rhodobacter spp.). Another way of avoiding the limitations set by available membranes is to produce the membrane protein as inclusion bodies. This is usually not desired but may allow preparation of active protein through solubilization of the inclusion bodies using denaturants followed by refolding. Successful refolding of β-barrel membrane proteins from inclusion bodies has been achieved but refolding of α-helical membrane proteins is an even greater challenge. For a separate discussion on inclusion bodies, see Inclusion bodies.

A modest growth and expression rate is beneficial to avoid the formation of inclusion bodies when using E. coli as a host. This can be achieved by the use of a weak promoter, a low concentration of inducer and/or lowering the growth temperature after induction.

An overview of different expression systems for membrane proteins is provided in Table 1.1.

Table 1.1. Overexpression systems used for prokaryotic and eukaryotic membrane protein production

Expression system Advantages Disadvantages
E. coli The most widely used overexpression system for (prokaryotic) membrane protein production. Often not suitable for overexpression of eukaryotic membrane proteins No glycosylation and limited posttranslational modifications
Yeast Can perform some posttranslational modifications Several different yeast systems have been used for membrane protein production Does not produce high cell densities (S. cerevisiae) Hyperglycosylation can occur   (S. cerevisiae) Different lipids (compared with mammalian cells)
Insect cells Less complex growth conditions compared with mammalian cells Relatively high expression levels Glycosylation More costly and complex than E. coli or yeast; different lipids (compared with mammalian cells)
Mammalian cells CHO, BHK and other cell lines are often used for functional studies of receptors Authentic (mammalian) protein is produced High cost and complex work
Rhodobacter spp. High expression levels through coordinated synthesis of foreign membrane proteins with synthesis of new internal membranes Different lipids (compared with mammalian cells)
Cell free Allows expression of toxic proteins and proteins that are easily degraded in vivo Allows incorporation of labelled and non-natural amino acids. High cost   Membrane protein insertion in membrane or detergent micelle has not been fully developed

Disposable 96-well filter plates, from GE Healthcare, prepacked with affinity purification media for histidine- or GST-tagged proteins can be used for reproducible, high-throughput screening of protein expression. Typical applications include expression screening of different constructs, screening for suitable detergents and solubility of proteins, and optimization of the conditions for small-scale parallel purification. Plates are available prepacked with Ni SepharoseTM High Performance or Ni Sepharose 6 Fast Flow for working with histidine-tagged proteins (His MultiTrapTM HP or His MultiTrap FF, respectively); and Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B for working with GST-tagged proteins (GST MultiTrap FF or GST MultiTrap 4B, respectively).