International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 3.2, pp. 92-93

Section 3.2.2. A consensus strategy for membrane-protein expression

J. A. Ernst,a,b D. G. Yansurac and C. M. Kothd*

aDepartment of Protein Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, USA,bDepartment of Protein Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA,cDepartment of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA, and dDepartment of Structural Biology, Genentech, 1 DNA Way, South San Francisco, California 94080, USA
Correspondence e-mail:  koth.christopher@gene.com

3.2.2. A consensus strategy for membrane-protein expression

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Historically, the likelihood of successful structure determination has been high for those membrane proteins that can be isolated from readily available abundant natural sources (Sakai & Tsukihara, 1998[link]). Unfortunately, most membrane proteins do not meet this criterion. In reality, the vast majority of high-resolution membrane-protein structures are of prokaryotic targets, expressed in Escherichia coli by recombinant methods (Willis & Koth, 2008[link]; Newby et al., 2009[link]). The reasons for this are simple. Attempting recombinant expression in E. coli is inexpensive, flexible, simple and easily scaled-up, and many constructs and strains can be screened quickly. As with crystallographic efforts for many soluble proteins (Gräslund et al., 2008[link]), most successful membrane-protein endeavours also commonly screen multiple constructs and/or orthologues for any given target (see, for example, Chang et al., 1998[link]; Doyle et al., 1998[link]), since the greater the number of unique constructs screened, the greater the chance that one will be successfully isolated and crystallized. Given the overwhelming use of E. coli for successful membrane-protein structures, a `first-pass' expression strategy suitable for almost any membrane protein is clear: attempt expression in E. coli and, whenever possible, screen multiple constructs, orthologues and strains.

3.2.2.1. Choosing the expression system and affinity tags

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In addition to the choice of expression host, one must also consider the expression system and the type and placement of affinity tags or fusion proteins. For E. coli, IPTG-inducible T7 polymerase-driven expression systems, such as those based on pET vectors (Studier & Moffatt, 1986[link]) and λDE3 lysogen strains, are the most widely used for membrane proteins, as is the case for soluble targets. Also, it has generally been observed that for most targets, protein expression is optimal at lower temperatures (i.e. <20 °C; Christendat et al., 2000[link]; Wang et al., 2003[link]; Dobrovetsky et al., 2005[link]). When structural studies are the desired outcome, the most common tagging strategy for membrane proteins is to engineer a stretch of at least six histidine residues at the amino or carboxyl terminus of the target constructs; this is used for >80% of successful targets (Willis & Koth, 2008[link]). If expression levels are sufficient (i.e. ≥0.05 mg g−1 cell paste), this often permits purification using a general two-step procedure: capture of the tagged protein by immobilized metal affinity chromatography (IMAC), followed by size-exclusion chromatography (SEC) (see below). Remarkably, this basic approach has proved successful for the crystallization of many membrane proteins, as discussed in the following section. If premature termination is observed during expression, the engineering of a carboxyl-terminal tag will ensure that these proteins are not isolated during purification. Also, extending the stretch of histidines to greater than six residues can improve the retention of membrane proteins on immobilized metal affinity resins. This can prove particularly useful, given the modest expression levels and reduced chromatographic resolution and recovery of many membrane targets (Dobrovetsky et al., 2005[link]; Eshaghi et al., 2005[link]; Surade et al., 2006[link]; Lewinson et al., 2008[link]). Other affinity tags are viable options, but are more rarely used. For example, although fusion proteins such as glutathione S-transferase (GST) and thioredoxin are widely used to promote expression and/or simplify the purification of soluble proteins [for an extensive comparison of various affinity tags, see Lichty et al. (2005[link])], they are rarely cited in expression strategies for polytopic membrane proteins.

3.2.2.2. Strategies for improving membrane-protein expression

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Low expression levels are common for many membrane proteins. Before testing alternative hosts, it may be worthwhile first to screen alternative E. coli expression systems, such as those using the tightly controlled arabinose promoter (Guzman et al., 1995[link]). Also, other E. coli strains can be tested. For example, the mutant C41 and C43 `Walker' expression strains (Miroux & Walker, 1996[link]; Wagner et al., 2008[link]) yield higher expression levels for some membrane proteins. Table 3.2.2.1[link] summarizes common strategies for improving the expression of membrane proteins in E. coli. If reasonable efforts in E. coli still do not yield sufficient expression, then an alternative expression host should be considered. The next most successful is yeast. Of the 22 recombinant eukaryotic membrane proteins whose structures have been solved by crystallography, half were produced using Pichia pastoris (seven targets), Saccharomyces cerevisiae (three targets) or Saccharomyces pombe (one target) (Raman et al., 2006[link]). Membrane proteins expressed in yeast are typically fused to an amino-terminal host signal sequence to promote proper mem­brane targeting. The use of higher eukaryotic hosts, such as insect and mammalian cells, is also increasing as more groups attempt to crystallize recombinant eukaryotic targets. The recent high-resolution structures of several G-protein-coupled receptors are notable examples of targets expressed successfully in insect cells (Cherezov et al., 2007[link]; Rasmussen et al., 2007[link]; Hanson et al., 2008[link]; Jaakola et al., 2008[link]; Warne et al., 2008[link]).

Table 3.2.2.1| top | pdf |
Strategies for improving recombinant membrane-protein expression in E. coli

Troubleshooting expression problems: if the `first-pass' expression strategy is unsuccessful, then some changes in the expression construct, host strain or induction conditions may be helpful. The symptoms of the problem often give a clue as to how to make these corrections.

Symptom Solution
E. coli colonies small after transformation into expression strain Need tighter promoter such as phoA, araC etc.
   
Unprocessed signal sequence If none is used in native situation, remove from construct
  If one is present in native situation, use an expression host co-translational signal sequence
  If still unprocessed, lower translation initiation strength
   
Mostly high-molecular-weight aggregates Decrease induction time
   
Rapid cell growth after induction with low expression levels Increase translation initiation strength
   
Poor cell growth after induction with low expression levels  
 (a) Accumulation maximum after only 1–3 h Decrease translation initiation rates or plasmid copy number
 (b) Accumulation increases out to 8–10 h post induction Usually successful:
   (a) Re-synthesize the gene, removing rare codons and optimizing codon pairs
   (b) Convert transmembrane and intracellular domain cysteines (normally reduced) to serines
   (c) Try different non-DE3 lysogen strains after switching to a non-T7 promoter
    (i) Cytoplasmic protease deletions
    (ii) Periplasmic protease deletions
    (iii) Heat-shock htpRTS
  Sometimes successful:
   (a) Try different C-terminal tags such as poly-his, Flag etc.
   (b) Try different temperatures of induction such as 20, 25, 30 °C
These methods are also useful for optimizing eukaryotic gene expression.

To date, there is only one report of a structure determined from cell-free synthesis of the target membrane protein (EmrE; Chen et al., 2007[link]). However, for several reasons, cell-free approaches should be explored as a complement to traditional expression methods. Firstly, the absence of a host cell negates toxicity problems for some target proteins. Secondly, many targets that do not express in cells can be produced cell-free (Ishihara et al., 2005[link]; Klammt et al., 2007[link]; Savage et al., 2007[link]). Thirdly, these systems allow for considerable flexibility with respect to reaction conditions and components. Cell-free systems for prokaryotic and eukaryotic hosts have been described (Schwarz et al., 2008[link]), some of which function in the presence of solubilizing detergents (Ishihara et al., 2005[link]; Klammt et al., 2007[link]; Savage et al., 2007[link]) or lipids (Kuruma et al., 2005[link]; Schwarz et al., 2007[link]), facilitating proper folding and downstream purification of membrane-protein targets.

Many eukaryotic proteins have undergone extensive construct engineering to improve expression and/or crystallization outcomes. These include fusion to T4 lysozyme, mutation of glycosylation sites, deletion of potentially disordered regions and/or targeted evolution to identify conformationally stable mutants (Long et al., 2005[link]; Cherezov et al., 2007[link]; Rosenbaum et al., 2007[link]; Jaakola et al., 2008[link]; Magnani et al., 2008[link]; Serrano-Vega et al., 2008[link]; Warne et al., 2009[link]). Also worth mentioning is the observation that incorporation of an amino-terminal `rhodopsin tag' comprising the first 20 amino acids of bovine rhodopsin (amino-acid sequence MNGTEGPNFYVPFSNKTGVV) has been found to boost dramatically the expression levels of eukaryotic membrane targets expressed in insect and mammalian cells (Krautwurst et al., 1998[link]).

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