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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    • "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.
    Metabolic Engineering 05/2012; 14(5):591-602. DOI:10.1016/j.ymben.2012.05.001 · 6.77 Impact Factor
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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.
    Microbial Cell Factories 05/2011; 10(1):37. DOI:10.1186/1475-2859-10-37 · 4.22 Impact Factor
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    • "Transmembrane proteins can be particularly difficult to successfully express in heterologous hosts (Freigassner et al., 2009). Quite often such proteins are poorly directed to the membrane and often are toxic to the cell (Luo et al., 2009; Steffensen and Pedersen, 2006; Wagner et al., 2006, 2008). "
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    ABSTRACT: DNA sequences are now far more readily available in silico than as physical DNA. De novo gene synthesis is an increasingly cost-effective method for building genetic constructs, and effectively removes the constraint of basing constructs on extant sequences. This allows scientists and engineers to experimentally test their hypotheses relating sequence to function. Molecular biologists, and now synthetic biologists, are characterizing and cataloging genetic elements with specific functions, aiming to combine them to perform complex functions. However, the most common purpose of synthetic genes is for the expression of an encoded protein. The huge number of different proteins makes it impossible to characterize and catalog each functional gene. Instead, it is necessary to abstract design principles from experimental data: data that can be generated by making predictions followed by synthesizing sequences to test those predictions. Because of the degeneracy of the genetic code, design of gene sequences to encode proteins is a high-dimensional problem, so there is no single simple formula to guarantee success. Nevertheless, there are several straightforward steps that can be taken to greatly increase the probability that a designed sequence will result in expression of the encoded protein. In this chapter, we discuss gene sequence parameters that are important for protein expression. We also describe algorithms for optimizing these parameters, and troubleshooting procedures that can be helpful when initial attempts fail. Finally, we show how many of these methods can be accomplished using the synthetic biology software tool Gene Designer.
    Methods in Enzymology, 489 edited by Christopher Voigt, 01/2011: chapter 3: pages 43-66; Elsevier Inc.., ISBN: 978-0123851208
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