| Budget Amount *help |
¥13,200,000 (Direct Cost: ¥13,200,000)
Fiscal Year 2001: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 2000: ¥8,100,000 (Direct Cost: ¥8,100,000)
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| Research Abstract |
The cell surface is a functional interface between the inside and the outside of cells. Surface proteins are responsible for most cell surface functions, serving as cell-cell adhesion molecules, specific receptors, enzymes, and transport proteins. Cells have systems for anchoring surface-specific proteins and for confining surface proteins to particular domains in the cell surface. From the viewpoint of application, the cell surface should be exploited by making use of the known transport mechanisms of proteins to the cell surface. Establishment of these systems to display heterologous proteins on the cell surface of microorganisms is expected to be useful for the segregation of produced polypeptides and the production of microbial biocatalysts, whole-cell adsorbents, and live vaccines. Utilization of the cell surface of living cells is also attractive for many applications in microbiology and molecular biology. Novel yeast cells armed with biocatalysts - glucoamylase, α-amylase, CM-ce
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llulase, β-glucosidase, and lipase - were constructed by a cell surface engineering system of yeast Saccharomyces cerevisiae. These surface-engineered yeast cells were termed "Arming yeasts ".
The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half of yeast α-agglutinin. Cell surface display of the functional proteins and peptides by cell surface engineering gives new functions to yeast cells. As hexa-His is a peptide well known as a chelator of divalent heavy metal ions, the display of the peptide as one of models of epitopes on the S.cerevisiae cell surface was tried. This cell surface-engineered yeast adsorbed three to eight times more copper ion than the parent strain. It was possible to recover about a half of copper ion adsorbed by whole cells with EDTA treatment without disintegrating cells. Furthermore, this engineered yeast would show copper ion tolerance, suggesting a novel breeding of the yeast with tolerance to toxic contaminants. Furthermore, for quantification of the amounts of proteins and peptides displaying on the yeast cell surface, a novel yeast strain displaying green fluorescent protein (GFP) from Aequorea victoria on its cell surface was constructed. This cell surface-engineering yeast strain emitted green fluorescence from the cell surface was useful to measure the number of molecules displaying on the cell surface and to monitor the concentrations of intra- and/or extracellular conditions by the various promoters. The S. cerevisiae strains exploited are the first examples of surface-engineered yeasts in which active proteins targeted to the cell surface endowed the cells with new beneficial properties. Less
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