Functional expression of mammalian receptors and membrane channels in different cells

In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.     

Quality Control in Eukaryotic Membrane Protein Overproduction

The overexpression of authentically folded eukaryotic membrane proteins in milligramme quantities is a fundamental prerequisite for structural studies. One of the most commonly used expression systems for the production of mammalian membrane proteins is the baculovirus expression system in insect cells. However, a detailed analysis by radioligand binding and comparative Western blotting of G protein-coupled receptors and a transporter produced in insect cells showed that a considerable proportion of the expressed protein was misfolded and incapable of ligand binding. In contrast, production of the same membrane proteins in stable inducible mammalian cell lines suggested that the majority was folded correctly. It was noted that detergent solubilisation of the misfolded membrane proteins using either digitonin or dodecylmaltoside was considerably less efficient than using sodium dodecyl sulfate or foscholine-12, whilst these detergents were equally efficient at solubilising correctly folded membrane proteins. This provides a simple and rapid test to suggest whether heterologously expressed mammalian membrane proteins are indeed correctly folded, without requiring radioligand binding assays. This will greatly facilitate the high-throughput production of fully functional membrane proteins for structural studies.     

NMR structures of polytopic integral membrane proteins

Abstract

Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all high-resolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both β-barrel and α-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design.     

The recombinant expression systems for structure determination of eukaryotic membrane proteins

Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryotic membrane protein structures have been obtained due to the technical challenges in the generation of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.     

Overexpression of mammalian integral membrane proteins for structural studies

Recent successes in the determination of atomic resolution structures of integral membrane proteins have relied on purifying the proteins from abundant natural sources. In contrast, the majority of mammalian receptors, ion channels and transporters need to be overexpressed to obtain sufficient material for structural studies. This has often proved to be very difficult. Overexpression studies on a wide range of mammalian membrane proteins have shown that a few can be expressed functionally in bacteria, but many others require an insect or mammalian cell host for activity or high level expression. The serotonin transporter, which has been expressed in all the major hosts available, is a good example that has given insights into the problem of overexpressing mammalian membrane proteins for structural studies.     

Production of membrane proteins using cell–free expression systems

Different overexpression systems are widely used in the laboratory to produce proteins in a reasonable amount for functional and structural studies. However, to optimize these systems without modifying the cellular functions of the living organism remains a challenging task. Cell-free expression systems have become a convenient method for the high-throughput expression of recombinant proteins, and great effort has been focused on generating high yields of proteins. Furthermore, these systems represent an attractive alternative for producing difficult-to-express proteins, such as membrane proteins. In this review, we highlight the recent improvements of these cell-free expression systems and their direct applications in the fields of membrane proteins production, protein therapy and modern proteomics.     

Production of membrane proteins using cell-free expression systems

Different overexpression systems are widely used in the laboratory to produce proteins in a reasonable amount for functional and structural studies. However, to optimize these systems without modifying the cellular functions of the living organism remains a challenging task. Cell-free expression systems have become a convenient method for the high-throughput expression of recombinant proteins, and great effort has been focused on generating high yields of proteins. Furthermore, these systems represent an attractive alternative for producing difficult-to-express proteins, such as membrane proteins. In this review, we highlight the recent improvements of these cell-free expression systems and their direct applications in the fields of membrane proteins production, protein therapy and modern proteomics.     

Characteristics affecting expression and solubilization of yeast membrane proteins

Biochemical and structural analysis of membrane proteins often critically depends on the ability to overexpress and solubilize them. To identify properties of eukaryotic membrane proteins that may be predictive of successful overexpression, we analyzed expression levels of the genomic complement of over 1000 predicted membrane proteins in a recently completed Saccharomyces cerevisiae protein expression library. We detected statistically significant positive and negative correlations between high membrane protein expression and protein properties such as size, overall hydrophobicity, number of transmembrane helices, and amino acid composition of transmembrane segments. Although expression levels of membrane and soluble proteins exhibited similar negative correlations with overall hydrophobicity, high-level membrane protein expression was positively correlated with the hydrophobicity of predicted transmembrane segments. To further characterize yeast membrane proteins as potential targets for structure determination, we tested the solubility of 122 of the highest expressed yeast membrane proteins in six commonly used detergents. Almost all the proteins tested could be solubilized using a small number of detergents. Solubility in some detergents depended on protein size, number of transmembrane segments, and hydrophobicity of predicted transmembrane segments. These results suggest that bioinformatic approaches may be capable of identifying membrane proteins that are most amenable to overexpression and detergent solubilization for structural and biochemical analyses. Bioinformatic approaches could also be used in the redesign of proteins that are not intrinsically well-adapted to such studies.     

Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression

Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.     

Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells.

The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line. UCP3 was expressed in relatively low levels (< 1 microg x mg(-1) membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins. Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in an incompetent conformation.     

Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells

It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks.     

Optimization of protein production in mammalian cells with a coexpressed fluorescent marker

The expression of mammalian proteins in sufficient abundance and quality for structural studies often presents formidable challenges. Many express poorly in bacterial systems, whereas it can be time consuming and expensive to produce them from cells of higher organisms. Here we describe a procedure for the direct selection of stable mammalian cell lines that express proteins of interest in high yield. Coexpression of a marker protein, such as green fluorescent protein, is linked to that of the desired protein through an internal ribosome entry site in the vector that is transfected into cells in culture. The coexpressed marker is used to select for highly expressing clonal cell lines. Applications are described to a membrane protein, the 5HT2c serotonin receptor, and to a secreted cysteine-rich protein, resistin. Besides providing an expeditious means for producing mammalian proteins for structural work, the resulting cell lines also readily support tests of functional properties and structure-inspired hypotheses.     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

Both Xenopus laevis oocytes and mammalian cells are widely used for heterologous expression of several classes of proteins, and membrane proteins especially, such as ion channels or receptors, have been extensively investigated in both cell types. A full characterization of a specific protein will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels.     

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems

Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.     

Enhanced Expression and Purification of Membrane Proteins by SUMO Fusion in Escherichia coli

Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein and 5-lipoxygenase-activating protein (FLAP) are among a large number of membrane proteins that are poorly expressed when traditional expression systems and methods are employed. Therefore to efficiently express difficult membrane proteins, molecular biologists will have to develop novel or innovative expression systems. To this end, we have expressed the SARS-CoV M and FLAP proteins in Escherichia coli by utilizing a novel gene fusion expression system that takes advantage of the natural chaperoning properties of the SUMO (small ubiquitin-related modifier) tag. These chaperoning properties facilitate proper protein folding, which enhances the solubility and biological activity of the purified protein. In addition to these advantages, we found that SUMO Protease 1, can cleave the SUMO fusion high specificity to generate native protein. Herein, we demonstrate that the expression of FLAP and SARS-CoV membrane proteins are greatly enhanced by SUMO fusions in E. coli.     

A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase α3β1 isoform

The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure–function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [³H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.     

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.