Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

Abstract

BackgroundThe 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.ResultsOverexpressed 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.ConclusionsInefficient 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.

Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae

“…We have analysed various protein spot parameters at two different experimental conditions: at standard 1x protein load (50 μg of whole cell lysate protein per gel, as recommended by manufacturer of IPG strips) and at high 2x protein load (100 μg of total protein per gel). General quantitative analysis of IPG- and NEPHGE-based 2DE methods is presented in Tables 1 and 2, respectively, whereas their “trial” comparison with the concrete biological experiment [17] is summarized in Table 3.…”

“…Here we performed a differential expression proteomics experiment using both methods and broad (pH 3–10) gradient range on UPR-Cyto stress in yeast Saccharomyces cerevisiae cells. The results were compared to our previous study of the same phenomenon using Invitrogen narrow range pH 4–7 IPG strips [17]. Our data suggest that NEPHGE-based 2DE method is a method of choice for the analysis of basic proteins.…”

General Introduction: Recombinant Protein Production and Purification of Insoluble Proteins

“…This can be exemplifi ed by the failing expression of virus surface glycoproteins (namely, mumps or measles hemagglutinin [ 102 ]) that renders inactive aggregates.…”

Heterologous Expression and Purification of ? Galactosidase Protein Using Affinity Chromatography

“…However, the GST-β galactosidase protein purified from the insoluble fraction was not sufficient for SDS-PAGE gel and western blotting analysis. Several trials following different protocols [15,25] to solubilize and purify the GST-β gal from inclusion bodies were carried out using lysis buffers containing DTT, Triton-X (1%), N-lauroylsarcosine sodium salt (sarkosyl) or NaCl (1M), but they were not successful. A likely reason for these results is that the GST tag increases solubility of β-galactosidase.…”

“…The recovery of both forms of proteins requires the disruption of the yeast cells as an initial step in the extraction and purification of proteins. Some of the examples in which direct intracellular expression have been successfully used to express heterologous gene products include the hepatitis B virus surface antigen (HBsAg) [7], human superoxide dismutase (hSOD) [8], fibroblast growth factor [9], human immunodeficiency virus type-1 (HIV1) env and gag polypeptides [10,11], surface antigens of malaria parasites [12], rat cytochrome C [13], α1-antitrypsin proteinase inhibitor [14] and human viral surface glycoproteins [15]. Considering all the benefits and advantages as like eukaryotic system, yeast was selected for heterologous expression of protein.…”