Tuning microbial hosts for membrane protein production. Microb Cell Fact 8:69

Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
Microbial Cell Factories (Impact Factor: 4.22). 12/2009; 8(1):69. DOI: 10.1186/1475-2859-8-69
Source: PubMed


The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months.
This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production.

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Available from: Anton Glieder
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    • "Because membrane proteins are found in their natural cellular environments in quantities that are too low for biochemical analysis, they have to be prepared by expression in heterologous hosts (Link and Georgiou, 2007; Wagner et al., 2006). Escherichia coli have been by far the most successful host for the preparative expression of heterologous membrane proteins (Freigassner et al., 2009; Wagner et al., 2006). Unfortunately, many mammalian membrane proteins fail to form correctly in bacteria and instead accumulate as cytoplasmic inclusion bodies that are very difficult to refold (Link and Georgiou, 2007). "
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    ABSTRACT: Low yields of recombinant expression represent a major barrier to the physical characterization of membrane proteins. Here, we have identified genes that globally enhance the production of properly folded G protein-coupled receptors (GPCRs) in Escherichia coli. Libraries of bacterial chromosomal fragments were screened using two separate systems that monitor: (i) elevated fluorescence conferred by enhanced expression of GPCR-GFP fusions and (ii) increased binding of fluorescent ligand in cells producing more active receptor. Three multi-copy hits were isolated by both methods: nagD, encoding the ribonucleotide phosphatase NagD; a fragment of nlpD, encoding a truncation of the predicted lipoprotein NlpD, and the three-gene cluster ptsN-yhbJ-npr, encoding three proteins of the nitrogen phosphotransferase system. Expression of these genes resulted in a 3- to 10-fold increase in the yields of different mammalian GPCRs. Our data is consistent with the hypothesis that the expression of these genes may serve to maintain the integrity of the bacterial periplasm and to provide a favorable environment for proper membrane protein folding, possibly by inducing a fine-tuned stress response and/or via modifying the composition of the bacterial cell envelope.
    Full-text · Article · May 2012 · Metabolic Engineering
    • "Prokaryotic expression systems, with Escherichia coli at the top, are among the most popular microbial systems for IMP expression[16]. However, differences in lipid bilayer composition and in the folding and secretion pathways, as well as the lack of post-translational modifications usually results in the failure of these systems to express eukaryotic IMPs or in their production at very low yields[16,17]. Among eukaryotic microbial systems, P. pastoris stands out as a successful expression host to obtain recombinant transmembrane α-helical proteins for which high-resolution structures have been successfully solved (Table 1). "
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    ABSTRACT: 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.
    No preview · Article · Jun 2011 · Biotechnology Journal
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    • "The characterization of such adverse conditions and the elicited cell responses have permitted a better understanding of the physiology and molecular biology of conformational stress [reviewed in [2]]. However, well-documented stress reactions in recombinant protein producing yeasts are limited mostly to unfolded protein response (UPR) in endoplasmic reticulum (ER) [2,3] and there is a lack of knowledge concerning the impact of other stress responses on heterologous membrane protein expression. Only recently two additional different stress responses induced by misfolded membrane proteins with lesions in a membrane span or a cytosolic domain (called UPR-M/C), and by misfolded cytosolic proteins that do not enter the secretory pathway at all (called UPR-Cyto) have been preliminarily characterized in Saccharomyces cerevisiae [4,5]. "
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    ABSTRACT: The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway.
    Full-text · Article · May 2011 · Microbial Cell Factories
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