Overexpression of membrane proteins from higher eukaryotes in yeasts

Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug–target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.     

Development of a high-throughput cloning strategy for characterization of Acinetobacter baumannii drug transporter proteins

Heterologous expression of membrane proteins in Escherichia coli often requires optimization to overcome problems with toxicity of the recombinant protein to the host cell. A number of Gateway-based destination vectors were constructed to investigate expression of membrane proteins using a high-throughput approach. These vectors were tested using putative drug transporter proteins from the multidrug and toxic compound extrusion (MATE) family and the resistance-nodulation-cell division superfamily encoded by the human pathogen Acinetobacter baumannii. Active transport of antibiotics and antiseptics mediated by efflux proteins contributes to the high level of multidrug resistance observed in A. baumannii. Substrates for 4 of the 5 putative efflux proteins investigated were identified using the expression vectors designed in this study. Additionally, a Gateway-based suicide vector was designed for construction of specific A. baumannii insertion disruption mutants. This knockout cloning strategy was tested and shown to be successful in inactivating AbeM4, a putative MATE family protein. Therefore, we have shown that the Gateway-based vectors constructed in this study are versatile tools that can be used for manipulation and characterization of membrane proteins.     

A novel method for viral display of ER membrane proteins on budded baculovirus

The baculovirus expression system has been used to express large quantities of various proteins, including membrane receptors. Here, we reveal a novel property of this expression system to be that certain membrane proteins can be displayed on the budded virus itself. We introduced the genes encoding sterol regulatory element-binding protein-2 (SREBP-2) or SREBP cleavage-activating protein (SCAP), important integral membrane proteins of the endoplasmic reticulum (ER) and/or the Golgi apparatus related to cellular cholesterol regulation, into a baculovirus vector. When insect cells were infected with SREBP-2 or SCAP recombinant viruses, it was found that these ER membrane proteins appeared on the budded baculovirus in addition to the host cell membrane fraction. Compared to proteins expressed on the cell membrane, membrane proteins displayed on virus exhibited both less aggregation and less degradation upon immunoblotting. Using this viral displayed SCAP as the screening antigen, we then generated a new monoclonal antibody specific against SCAP, which was useful for immunological localization studies. This system, which takes advantage of the viral display of membrane proteins, should prove to be a powerful additional tool for postgenomic protein analysis.     

EspA filament-mediated protein translocation into red blood cells

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro . As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria–RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria–RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.     

The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.     

Alphaviruses as tools in neurobiology and gene therapy

The broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for Semliki Forest virus (SFV) and Sindbis virus (SIN). The generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. Despite the viral cytopathogenic effects, functional assays on recombinant proteins are possible for a time-period of at least 24 hours post-infection. The high percentage (80-95%) of primary neurons infected with SFV has allowed localization and functional studies of recombinant proteins in these primary cell cultures. Through multiple infection studies the interaction of receptor and G protein subunits has become feasible. Establishment of efficient scale-up procedures has allowed production of large quantities of recombinant protein. Potential gene therapy applications of alphaviruses could be demonstrated by injection of recombinant SIN particles expressing beta-galactosidase into mouse brain. Tissue/cell specific infection has been achieved by introduction of an IgG-binding domain of protein A domain into one of the spike proteins of SIN. This enabled efficient targeting of infection to human lymphoblastoid cells.     

Prospects for vaccines of helminth parasites of grazing ruminants

Defined molecular vaccines for several ruminant heliminth parasites are being pursued at several different laboratories. The most fruitful sources of antigen have been oncosphere surface proteins, excretory/secretory products and integral gut membrane proteins. Nematode gut membrane proteins are unconventional in that they do not come into contact with the host immune response during infection, a feature which brings advantages as well as disadvantages. The genes encoding several of the protective antigens have been cloned, but only in the case of the oncosphere surface proteins has substantial protection been reported with recombinant versions. In addition to the problem of identifying suitable expression systems, issues such as choice of adjuvant and/or the possible use of a vaccine vector have to be solved before molecular vaccines for the economically important helminths can be launched. Of the latter, it seems that vaccines for Haemonchus and Fasciola are the brightest prospects.     

Import of bacterial pathogenicity factors into mitochondria

Recent research on the mechanism underlying the interaction of bacterial pathogens with their host has shifted the focus to secreted microbial proteins affecting the physiology and innate immune response of the target cell. These proteins either traverse the plasma membrane via specific entry pathways involving host cell receptors or are directly injected via bacterial secretion systems into the host cell, where they frequently target mitochondria. The import routes of bacterial proteins are mostly unknown, whereas the effect of mitochondrial targeting by these proteins has been investigated in detail. For a number of them, classical leader sequences recognized by the mitochondrial protein import machinery have been identified. Bacterial outer membrane beta-barrel proteins can also be recognized and imported by mitochondrial transporters. Besides an obvious importance in pathogenicity, understanding import of bacterial proteins into mitochondria has a highly relevant evolutionary aspect, considering the endosymbiotic, proteobacterial origin of mitochondria. The review covers the current knowledge on the mitochondrial targeting and import of bacterial pathogenicity factors.     

提高大肠杆菌可溶性重组蛋白表达产率的研究进展

大肠杆菌表达重组蛋白相比真核细胞具有成本低廉、大规模发酵容易、条件易于自动化控制等优点,通过大肠杆菌表达重组蛋白是一种高效、经济的途径,重组蛋白表达量可达到大肠杆菌总蛋白质量的50%。具有正常生化活性的重组蛋白通常为可溶性形式,因而对于以得到活性产物(如抗体、酶等)为目的的研究,通常采用可溶性表达途径。目前已有多种以可溶性重组蛋白为活性物质的治疗性药物经批准上市,但并非所有外源基因均能实现可溶性高表达,因此重组蛋白的可溶性高表达具有重要研究价值。在总结近年提高经大肠杆菌可溶性表达重组蛋白产率研究的基础上,从启动子的选择、SD序列的引入、信号肽的优化、宿主细胞的选择、共表达其他蛋白质,高密度发酵等方面阐释在大肠杆菌中提高可溶性重组蛋白表达产率的方法。     

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.     

Modulating secretory pathway pH by proton channel co‐expression can increase recombinant protein stability in plants

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host‐cell engineering approaches are being developed to ameliorate host‐cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid‐susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co‐expression. The co‐expression of a heterologous ion channel to stabilize acid‐labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.     

Alphaviruses as Tools in Neurobiology and Gene Therapy

Abstract

The broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for Semliki Forest virus (SFV) and Sindbis virus (SIN). The generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. Despite the viral cytopathogenic effects, functional assays on recombinant proteins are possible for a time-period of at least 24 hours post-infection. The high percentage (80–95%) of primary neurons infected with SFV has allowed localization and functional studies of recombinant proteins in these primary cell cultures. Through multiple infection studies the interaction of receptor and G protein subunits has become feasible. Establishment of efficient scale-up procedures has allowed production of large quantities of recombinant protein. Potential gene therapy applications of alphaviruses could be demonstrated by injection of recombinant SIN particles expressing β-galactosidase into mouse brain. Tissue/cell specific infection has been achieved by introduction of an IgG-binding domain of protein A domain into one of the spike proteins of SIN. This enabled efficient targeting of infection to human lymphoblastoid cells.     

Interaction of heat shock protein 70 with membranes depends on the lipid environment

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.     

Protein expression in yeast; comparison of two expression strategies regarding protein maturation

The driving force for the modification of existing, or the development of new, protein expression systems lies in the identification of a tremendous number of potential novel drug targets through recent genomics approaches. Saccharomyces cerevisiae as a host for recombinant protein expression, offers many advantages, as its biosynthetic pathways resemble higher eukaryotic cells in many aspects. Two yeast vectors were compared to evaluate the versatility of this organism for expression of recombinant proteins. One expression vector enables the secretion of the recombinant protein into the culture medium through fusion with the leader sequence of the mating-type pheromone alpha; the other directs the expression product into the cytoplasm of the yeast cell through fusion with ubiquitin. To facilitate immunological detection and purification, proteins were expressed as fusions to an octapeptide, the so-called Flag-tag, which is recognised by a monoclonal antibody in the presence of Ca2+. We chose 20 functionally different cDNAs to compare the efficiency of both expression systems. All cDNAs could be expressed at the correct size but at varying yields and purity. Both expression systems differed greatly in the degree of glycosylation and other, not further analysed, post-translational modifications. Secretion of all model proteins into the cell culture supernatant could be accomplished if membrane domains or signal sequences were absent, but many proteins were heavily glycosylated as demonstrated by lectin mapping or enzymatical deglycosylation. Some proteins, however, were expressed as homogenous products, and could be easily purified for further functional studies. Further investigations on the expression biology of yeast are required, in order to optimise the conditions of fermentation which may finally lead to more homogeneous expression products.     

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.     

Advances in the production of membrane proteins in Pichia pastoris

Membrane proteins play key roles in diverse cellular functions and have become the target for a large number of pharmacological drugs. Despite representing about 20-30% of cellular proteins, their characterization is long overdue since they are difficult to handle, to purify from their natural source or to obtain as recombinant proteins. Pichia pastoris is a methylotrophic yeast species increasingly used as a host for heterologous protein expression for both research and industrial purposes. Over the past few years many efforts have allowed important advances in the development of this expression system for the expression and production of membrane proteins. The most recent achievements in improving yield and proper folding of integral membrane proteins are summarized in this review.     

Apolipoproteins L1-6 share key cation channel-regulating residues but have different membrane insertion and ion conductance properties

The human apolipoprotein L gene family encodes the apolipoprotein L1–6 (APOL1–6) proteins, which are effectors of the innate immune response to viruses, bacteria and protozoan parasites. Due to a high degree of similarity between APOL proteins, it is often assumed that they have similar functions to APOL1, which forms cation channels in planar lipid bilayers and membranes resulting in cytolytic activity. However, the channel properties of the remaining APOL proteins have not been reported. Here, we used transient overexpression and a planar lipid bilayer system to study the function of APOL proteins. By measuring lactate dehydrogenase release, we found that APOL1, APOL3, and APOL6 were cytolytic, whereas APOL2, APOL4, and APOL5 were not. Cells expressing APOL1 or APOL3, but not APOL6, developed a distinctive swollen morphology. In planar lipid bilayers, recombinant APOL1 and APOL2 required an acidic environment for the insertion of each protein into the membrane bilayer to form an ion conductance channel. In contrast, recombinant APOL3, APOL4, and APOL5 readily inserted into bilayers to form ion conductance at neutral pH, but required a positive voltage on the side of insertion. Despite these differences in membrane insertion properties, the ion conductances formed by APOL1-4 were similarly pH-dependent and cation-selective, consistent with conservation of the pore-lining region in each protein. Thus, despite structural conservation, the APOL proteins are functionally different. We propose that these proteins interact with different membranes and under different voltage and pH conditions within a cell to effect innate immunity to different microbial pathogens.     

The import competence of a peroxisomal membrane protein is determined by Pex19p before the docking step

Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.     

A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.