Expression Of Mammalian Proteins Research Articles

Expression of mammalian proteins for diagnostics and therapeutics: a review.

BackgroundAntibodies have proven to be remarkably successful for biomedical applications. They play important roles in epidemiology and medicine from diagnostics of diseases to therapeutics, treating diseases from incessant chronic diseases such as rheumatology to pandemic outbreaks. With no end in sight for the demand for antibody products, optimizations and new techniques must be expanded to accommodate this.Methods and ResultsThis review discusses optimizations and techniques for antibody production through choice of discovery platforms, expression systems, cell culture mediums, and other strategies to increase expression yield. Each system has its own merits and demerits, and the strategy chosen is critical in addressing various biological aspects.ConclusionsThere is still insufficient evidence to validate the efficacy of some of these techniques, and further research is needed to consolidate these industrial production systems. There is no doubt that more strategies, systems, and pipelines will contribute to enhance biopharmaceutical production.

Peptide tags and domains for expression and detection of mammalian membrane proteins at the cell surface

Transport proteins mediating solute transfer across the plasma membrane constitute an important group of cell-surface proteins. There are several diseases resulting from mutations in these proteins that interfere with their transport function or trafficking, depending on the impact of the mutations on protein folding and structure. Recent advances in successful treatment of some of these diseases with small molecules which correct the mutations-induced folding and structural changes underline the need for detailed structural and biophysical characterization of membrane proteins.

Peptide Tags and Domains for Expression and Detection of Mammalian Membrane Proteins at the Cell Surface.

Normal functions of cell-surface proteins are dependent on their proper trafficking from the site of synthesis to the cell surface. Transport proteins mediating solute transfer across the plasma membrane constitute an important group of cell-surface proteins. There are several diseases resulting from mutations in these proteins that interfere with their transport function or trafficking, depending on the impact of the mutations on protein folding and structure. Recent advances in successful treatment of some of these diseases with small molecules which correct the mutations-induced folding and structural changes underline the need for detailed structural and biophysical characterization of membrane proteins. This requires methods to express and purify these proteins using heterologous expression systems. Here, using the solute carrier (SLC) transporter NaCT (Na+-coupled citrate transporter) as an example, we describe experimental strategies for this approach. We chose this example because several mutations in NaCT, distributed throughout the protein, cause a severe neurologic disease known as early infantile epileptic encephalopathy-25 (EIEE-25). NaCT was modified with various peptide tags, including a RGS-His10, a Twin-Strep, the SUMOstar domain, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. When transiently expressed in HEK293 cells, recombinant NaCT proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited citrate transport activity similar to the nontagged protein. Surface NaCT expression was enhanced by the presence of SUMOstar on the N-terminus. The dual-purpose peptide epitopes RGS-His10 and Twin-Strep facilitated detection of NaCT by immunohistochemistry and western blot and may serve useful tags for affinity purification. This approach sets the stage for future analyses of mutant NaCT proteins that may alter protein folding and trafficking. It also demonstrates the capability of a transient mammalian cell expression system to produce human NaCT of sufficient quality and quantity to augment future biophysical and structural studies and drug discovery efforts.

DNA Transfer into Animal Cells Using Stearylated CPP Based Transfection Reagent.

Mammalian transient expression systems enable flexible and rapid production of proteins. They're ideal for expression of human or other mammalian proteins because these systems generate recombinant proteins with more native folding and post-translational modifications-such as glycosylation-than expression systems based on hosts such as E. coli, yeast, or insect cells.Here we describe transient protein production using QMCF Technology (Icosagen, Estonia) that uses specific suspension-adapted mammalian cell lines (QMCF cells), appropriate QMCF episomal expression vector, a chemically defined animal origin-free, serum-free medium, and Reagent 007™ (Icosagen, Estonia) for efficient delivery of nucleic acids for transfection of mammalian cells in comparison with PEI MAX™ (Transfection Grade Linear Polyethylenimine Hydrochloride, Polysciences).

Recombinant Yarrowia lipolytica strains for the heterologous expression of multi-component enzyme systems: Expression of mammalian steroidogenic proteins

New Yarrowia lipolytica strains for the co-expression of steroidogenic mammalian proteins were obtained in this study. For this purpose, a two-step approach for constructing recombinant strains that permits the simple introduction of several expression cassettes encoding heterologous proteins into the yeast genome was successfully applied. This study tested two series of integrative multi-copy expression vectors containing cDNAs for the mature forms of P450scc system components (cytochrome P450scc (CYP11A1), adrenodoxin reductase, adrenodoxin, or fused adrenodoxin-P450scc) or for P45017α (CYP17A1) under the control of the isocitrate lyase promoter pICL1, which were constructed using the basic plasmids p64PT or p67PT (rDNA or the long terminal repeat (LTR) zeta of Ylt1 as integration targeting sequences and ura3d4 as a multi-copy selection marker). This study demonstrated the integration of up to three expression vectors containing different heterologous cDNA via their simultaneous transformation into haploid recipient strains. Additionally, further combinations of the different expression cassettes in one strain were obtained by subsequent diploidisation using selected haploid multi-copy transformants. Thus, recombinant strains containing three to five different expression cassettes were obtained, as demonstrated by Southern blotting. Expression of P450scc system proteins was identified by western blotting. The presented method for recombinant strain construction is a useful tool for the heterologous expression of multi-component enzyme systems in Y. lipolytica.

Predicted N-terminal N-linked glycosylation sites may underlie membrane protein expression patterns in Saccharomyces cerevisiae.

N-linked glycosylation is one type of posttranslational modification that proteins undergo during expression. The following describes the effects of N-linked glycosylation on high-level membrane protein expression in yeast with an emphasis on Saccharomyces cerevisiae. N-linked glycosylation is highlighted here as an important consideration when preparing membrane protein gene constructs for expression in S. cerevisiae, which continues to be used as a workhorse in both research and industrial applications. Non-native N-linked glycosylation commonly occurs during the heterologous expression of mammalian proteins in many yeast species which can have important immunological consequences when used in the production of biotherapeutic proteins or peptides. Further, non-native N-linked glycosylation can lead to improper protein folding and premature degradation, which can impede high-level expression yields and hinder downstream analysis. Multiple strategies are presented in this article, which suggest different methods that can be implemented to circumvent the unwanted consequences of N-linked glycosylation during the expression process. These considerations may have long-term benefits for high-level protein production in S. cerevisiae across a broad spectrum of expression targets with special emphasis placed on G-protein coupled receptors, one of the largest families of membrane proteins.

Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.

The expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.

Identifying Anti-prion Chemical Compounds Using a Newly Established Yeast High-Throughput Screening System

Prion-like protein aggregation underlies the pathology of a group of fatal neurodegenerative diseases in humans, including Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis, and transmissible spongiform encephalopathy. At present, few high-throughput screening (HTS) systems are available for anti-prion small-molecule identification. Here we describe an innovative phenotypic HTS system in yeast that allows for efficient identification of chemical compounds that eliminate the yeast prion [SWI+]. We show that some identified anti-[SWI+] compounds can destabilize other non-[SWI+] prions, and their antagonizing effects can be prion- and/or variant specific. Intriguingly, among the identified hits are several previously identified anti-PrPSc compounds and a couple of US Food and Drug Administration-approved drugs for AD treatment, validating the efficacy of this HTS system. Moreover, a few hits can reduce proteotoxicity induced by expression of several pathogenic mammalian proteins. Thus, we have established a useful HTS system for identifying compounds that can potentially antagonize prionization and human proteinopathies.

Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host.

In this review, we summarize the current knowledge concerning the eukaryotic protozoan parasite Leishmania tarentolae, with a main focus on its potential for biotechnological applications. We will also discuss the genus, subgenus, and species-level classification of this parasite, its life cycle and geographical distribution, and similarities and differences to human-pathogenic species, as these aspects are relevant for the evaluation of biosafety aspects of L. tarentolae as host for recombinant DNA/protein applications. Studies indicate that strain LEM-125 but not strain TARII/UC of L. tarentolae might also be capable of infecting mammals, at least transiently. This could raise the question of whether the current biosafety level of this strain should be reevaluated. In addition, we will summarize the current state of biotechnological research involving L. tarentolae and explain why this eukaryotic parasite is an advantageous and promising human recombinant protein expression host. This summary includes overall biotechnological applications, insights into its protein expression machinery (especially on glycoprotein and antibody fragment expression), available expression vectors, cell culture conditions, and its potential as an immunotherapy agent for human leishmaniasis treatment. Furthermore, we will highlight useful online tools and, finally, discuss possible future applications such as the humanization of the glycosylation profile of L. tarentolae or the expression of mammalian recombinant proteins in amastigote-like cells of this species or in amastigotes of avirulent human-pathogenic Leishmania species.

Yeast Models for the Study of Amyloid-Associated Disorders and Development of Future Therapy.

First described almost two decades ago, the pioneering yeast models of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases, have become well-established research tools, providing both basic mechanistic insights as well as a platform for the development of therapeutic agents. These maladies are associated with the formation of aggregative amyloid protein structures showing common characteristics, such as the assembly of soluble oligomeric species, binding of indicative dyes, and apoptotic cytotoxicity. The canonical yeast models have recently been expanded by the establishment of a model for type II diabetes, a non-neurological amyloid-associated disease. While these model systems require the exogenous expression of mammalian proteins in yeast, an additional amyloid-associated disease model, comprising solely mutations of endogenous yeast genes, has been recently described. Mutated in the adenine salvage pathway, this yeast model exhibits adenine accumulation, thereby recapitulating adenine inborn error of metabolism disorders. Moreover, in line with the recent extension of the amyloid hypothesis to include metabolite amyloids, in addition to protein-associated ones, the intracellular assembly of adenine amyloid-like structures has been demonstrated using this yeast model. In this review, we describe currently available yeast models of diverse amyloid-associated disorders, as well as their impact on our understanding of disease mechanisms and contribution to future potential drug development.

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson's disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individualproteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Sialylation and sialyltransferase in insects.

Sialic acids are negatively charged nine carbon monosaccharides located terminally on glycoproteins and glycolipids that control cellular physiological processes. Sialylation is a post translational modification (ptm) regulated by enzymes and has been studied in prokaryotes including bacteria, dueterostomes including vertebrates, Cephalochordates, Ascidians, Echinoderms and protostomes including Molluscs and Arthropods and Plant. Although diverse structures of sialylated molecules have been reported in different organisms, unravelling sialylation in insect biology is a completely new domain. Within protostomes, the study of sialylation in members of Phylum Arthropoda and Class Insecta finds importance. Reports on sialylation in some insects exist. Genetically engineered components of sialylation pathway in Spodoptera frugiperda (Sf9) cell lines have enabled our understanding of sialylation and expression of mammalian proteins in insects. In this study we have summarised the finding on (i) sialylated molecules (ii) processes and enzymes involved (iii) function of sialylation (iv) genetic engineering approaches and generation of mammalian protein expression systems (v) a comparison of sialylation machinery in insects with that of mammals (vi) genes and transcriptional regulation in insects. At present no information on structural studies of insect sialyltransferase (STs) exist. We report minor differences in ST structure in insects on complete protein sequences recorded in Genbank through in silico approaches. An indepth study of all the components of the sialylation pathway in different insect species across different families and their evolutionary significance finds importance as the future scope of this review.

Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system.

Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense 1H-13C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective 13C incorporation by the P. pastoris expression system.

Improved high-yield expression, purification and refolding of recombinant mammalian prion proteins under aerosol-free elevated biological safety conditions

Production of recombinant prion proteins is of crucial relevance in food technology (analytical standards, assay development) but also in basic research, most importantly structural biology (NMR, X-ray diffraction). Structural approaches conveniently allow for sophisticated investigation of prion disease pathogenesis, but usually require large amounts of sample material. Recently, working with recombinant prion proteins has been recategorized to biosafety levels > S1 as infectious prions may readily be generated de novo and become airborne via aerosols. Heterologous expression should therefore be established with appropriately adjusted safety precautions. We have developed a protocol for high-yield expression, purification and refolding of recombinant mammalian prion proteins at elevated biological safety levels by introducing means of abolishing aerosol formation and propagation.

Orchestration of the mammalian host cell glucose transporter proteins-1 and 3 by Chlamydia contributes to intracellular growth and infectivity.

Chlamydia are gram-negative obligate intracellular bacteria that replicate within a discrete cellular vacuole, called an inclusion. Although it is known that Chlamydia require essential nutrients from host cells to support their intracellular growth, the molecular mechanisms for acquiring these macromolecules remain uncharacterized. In the present study, it was found that the expression of mammalian cell glucose transporter proteins 1 (GLUT1) and glucose transporter proteins 3 (GLUT3) were up-regulated during chlamydial infection. Up-regulation was dependent on bacterial protein synthesis and Chlamydia-induced MAPK kinase activation. GLUT1, but not GLUT3, was observed in close proximity to the inclusion membrane throughout the chlamydial developmental cycle. The proximity of GLUT1 to the inclusion was dependent on a brefeldin A-sensitive pathway. Knockdown of GLUT1 and GLUT3 with specific siRNA significantly impaired chlamydial development and infectivity. It was discovered that the GLUT1 protein was stabilized during infection by inhibition of host-dependent ubiquitination of GLUT1, and this effect was associated with the chlamydial deubiquitinase effector protein CT868. This report demonstrates that Chlamydia exploits host-derived transporter proteins altering their expression, turnover and localization. Consequently, host cell transporter proteins are manipulated during infection as a transport system to fulfill the carbon source requirements for Chlamydia.

TRPV4-dependent induction of a novel mammalian cold-inducible protein SRSF5 as well as CIRP and RBM3

Cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) are two evolutionarily conserved RNA-binding proteins that are structurally related to hnRNPs and upregulated in response to moderately low temperatures in mammalian cells. Although contributions of splicing efficiency, the gene promoters activated upon mild hypothermia and the transcription factor Sp1 to induction of CIRP have been reported, precise mechanisms by which hypothermia and other stresses induce the expression of mammalian cold-inducible proteins (CIPs) are poorly understood. By screening the serine/arginine-rich splicing factors (SRSFs), we report that the transcript and protein levels of SRSF5 were increased in mammalian cells cultured at 32 °C. Expression of SRSF5 as well as CIRP and RBM3 were also induced by DNA damage, hypoxia, cycloheximide and hypotonicity. Immunohistochemical studies demonstrated that SRSF5 was constitutively expressed in male germ cells and the level was decreased in human testicular germ cell tumors. SRSF5 facilitated production of p19 H-RAS, and increased sensitivity to doxorubicin in human U-2 OS cells. Induction of CIPs was dependent on transient receptor potential vanilloid 4 (TRPV4) channel protein, but seemed independent of its ion channel activity. These findings indicate a previously unappreciated role for the TRP protein in linking environmental stress to splicing.

Sequence determinants of the Caenhorhabditis elegans dopamine transporter dictating in vivo axonal export and synaptic localization

The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies. Loss of function mutations in the gene encoding the Caenhorhabditis elegans DAT (dat-1) produces rapid immobility when animals are placed in water, a phenotype termed swimming-induced paralysis (Swip). The ability of a DA neuron-expressed, GFP-tagged DAT-1 fusion protein (GFP::DAT-1) to localize to synapses and rescue Swip in these animals provides a facile approach to define sequences supporting DAT somatic export and function in vivo. In prior studies, we found that truncation of the last 25 amino acids of the DAT-1 C-terminus (Δ25) precludes Swip rescue, supported by a deficit in GFP::DAT-1 synaptic localization. Here, we further defined the elements within Δ25 required for DAT-1 export and function in vivo. We identified two conserved motifs (584KW585 and 591PYRKR595) where mutation results in a failure of GFP::DAT-1 to be efficiently exported to synapses and restore DAT-1 function. The 584KW585 motif conforms to a sequence proposed to support SEC24 binding, ER export from the endoplasmic reticulum (ER), and surface expression of mammalian DAT proteins, whereas the 591PYRKR595 sequence conforms to a 3R motif identified as a SEC24 binding site in vertebrate G-protein coupled receptors. Consistent with a potential role of SEC24 orthologs in DAT-1 export, we demonstrated DA neuron-specific expression of a sec-24.2 transcriptional reporter. Mutations of the orthologous C-terminal sequences in human DAT (hDAT) significantly reduced transporter surface expression and DA uptake, despite normal hDAT protein expression. Although, hDAT mutants retained SEC24 interactions, as defined in co-immunoprecipitation studies. However, these mutations disrupted the ability of SEC24D to enhance hDAT surface expression. Our studies document an essential role of conserved DAT C-terminal sequences in transporter somatic export and synaptic localization in vivo, that add further support for important roles for SEC24 family members in efficient transporter trafficking.

Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these “undesirable” residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.

Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.