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.     

Overexpression of membrane proteins in mammalian cells for structural studies

Abstract

The number of structures of integral membrane proteins from higher eukaryotes is steadily increasing due to a number of innovative protein engineering and crystallization strategies devised over the last few years. However, it is sobering to reflect that these structures represent only a tiny proportion of the total number of membrane proteins encoded by a mammalian genome. In addition, the structures determined to date are of the most tractable membrane proteins, i.e., those that are expressed functionally and to high levels in yeast or in insect cells using the baculovirus expression system. However, some membrane proteins that are expressed inefficiently in these systems can be produced at sufficiently high levels in mammalian cells to allow structure determination. Mammalian expression systems are an under-used resource in structural biology and represent an effective way to produce fully functional membrane proteins for structural studies. This review will discuss examples of vertebrate membrane protein overexpression in mammalian cells using a variety of viral, constitutive or inducible expression systems.     

Recent advances on proteins of plant terminal membranes.

Since the beginning of the 1990s, our knowledge of the protein equipment of plant membranes progresses at an accelerating pace, owing to the irruption of molecular biology tools and genetics strategies in plant biology. Map-based cloning strategies and exploration of EST databases rapidly enrich the catalog of cDNA or gene sequences expected to code for membrane proteins. The accumulation of 'putative' membrane proteins reinforces the need for structural, functional and physiological information. Indeed, ambiguities often exist concerning the association to a membrane, the membrane identity and the topology of the protein inserted in the membrane. The combination of directed mutagenesis and heterologous expression of plant genes in various systems and plant reverse genetics has opened the possibility to study molecular and physiological functions. This review will emphasize how these tools have been essential for the exciting recent discoveries on plant terminal membrane proteins. These discoveries concern a variety of transport systems for ions, organic solutes including auxin, water channels, a large collection of systems suspected to act as receptors of chemical signals, proteins thought to control vesicle trafficking and enzymatic systems.     

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.     

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.     

Cell-free expression as an emerging technique for the large scale production of integral membrane protein

Membrane proteins are highly underrepresented in structural data banks due to tremendous difficulties that occur upon approaching their structural analysis. Inefficient sample preparation from conventional cellular expression systems is in many cases the first major bottleneck. Preparative scale cell-free expression has now become an emerging alternative tool for the high level production of integral membrane proteins. Many toxic effects attributed to the overproduction of recombinant proteins are eliminated by cell-free expression as viable host cells are no longer required. A unique characteristic is the open nature of cell-free systems that offers a variety of options to manipulate the reaction conditions in order to protect or to stabilize the synthesized recombinant proteins. Detergents or lipids can easily be supplemented and membrane proteins can therefore be synthesized directly into a defined hydrophobic environment of choice that permits solubility and allows the functional folding of the proteins. Alternatively, cell-free produced precipitates of membrane proteins can efficiently be solubilized in mild detergents after expression. Highly valuable for structural approaches is the fast and efficient cell-free production of uniformly or specifically labeled proteins. A considerable number of membrane proteins from diverse families like prokaryotic small multidrug transporters or eukaryotic G-protein coupled receptors have been produced in cell-free systems in high amounts and in functionally active forms. We will give an overview about the current state of the art of this new approach with special emphasis on technical aspects as well as on the functional and structural characterization of cell-free produced membrane proteins.     

Production of crystallizable fragments of membrane proteins

Many membrane proteins feature autonomously folded extramembranous domains which, when isolated from the intact protein, perform biochemical functions relevant to biological activity. Whereas intact membrane proteins usually require detergent solubilization for purification, most extramembranous fragments are soluble in aqueous solution. If appropriately constructed, such fragments are often crystallizable and the resulting atomic structures can lead to important biological insight. In most instances, these fragments are produced in recombinant expression systems. To be crystallizable, molecular fragments should be uniform in composition and conformation and be available in abundance. Considerations for the production of crystallizable fragments of membrane proteins include the definition of fragment boundaries, the control of nonuniformities introduced by glycosylation or phosphorylation, and optimization of expression systems. These aspects are addressed here in general terms and in the case studies of applications to CD4, CD8, the insulin receptor kinase, and N-cadherin.     

Lactococcus lactis,an Alternative System for Functional Expression of Peripheral and Intrinsic Arabidopsis Membrane Proteins

Background

Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.

Methodology/Principal Findings

The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.

Conclusions/Significance

Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.     

Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors

In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.     

IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems

Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.     

Cell-free complements in vivo expression of the E. coli membrane proteome

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.     

Expression screening of integral membrane proteins from Helicobacter pylori 26695

The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins < or =40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions.     

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.     

Synthesis of membrane proteins in eukaryotic cell‐free systems

Cell‐free protein synthesis (CFPS) is a valuable method for the fast expression of difficult‐to‐express proteins as well as posttranslationally modified proteins. Since cell‐free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell‐free translation systems are capable to provide high molecular weight membrane proteins, fluorescent‐labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.     

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.     

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.     

Revolutionizing membrane protein overexpression in bacteria

The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory, especially for eukaryotic membrane proteins. This has initiated a revolution of membrane protein overexpression in bacteria. Recent studies have shown that it is feasible to (i) engineer or select for E. coli strains with strongly improved membrane protein overexpression characteristics, (ii) use bacteria other than E. coli for the expression of membrane proteins, (iii) engineer or select for membrane protein variants that retain functionality but express better than the wild-type protein, and (iv) express membrane proteins using E. coli-based cell-free systems.     

Bacterial expression systems for recombinant protein production: E. coli and beyond

Escherichia coli expression system continues to dominate the bacterial expression systems and remain to be the preferred system for laboratory investigations and initial development in commercial activities or as a useful benchmark for comparison among various expression platforms. Some new developments in overcoming its shortcomings are reviewed in this paper, including antibiotics-free selection plasmids, extracellular production, and posttranslational modifications. The ability for E. coli to make mg glycosylated proteins promises even broader applications of the E. coli system in the future. Significant progresses have also been made over the past few years in alternative bacterial expression systems. Notably, the Lactoccocus lactis system has proven to be a viable choice for membrane proteins. Additionally, several Pseudomonas systems were developed and achieved product titers comparable to E. coli systems. Other bacterial systems such as Streptomyces, coryneform bacteria, and halophilic bacteria offer advantages in some niche areas, providing more choices of bacterial expression systems for recalcitrant proteins.     

Breaking the bottleneck: eukaryotic membrane protein expression for high-resolution structural studies

The recombinant expression of eukaryotic membrane proteins has been a major stumbling block in efforts to determine their structures. In the last two years, however, five such proteins have yielded high-resolution X-ray or electron diffraction data, opening the prospect of increased throughput for eukaryotic membrane protein structure determination. Here, we summarize the major expression systems available, and highlight technical advances that should facilitate more systematic screening of expression conditions for this physiologically important class of targets.     

Strategies for prokaryotic expression of eukaryotic membrane proteins

High-level heterologous expression of integral membrane proteins at full-length is a useful tool for their structural and functional characterization. Here, systems that have previously been used for efficient bacterial expression of eukaryotic membrane proteins are reviewed and novel vectors consisting of a modular fusion moiety based on nuclease A from Staphylococcus aureus are presented.