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Development of epitope-displaying yeasts as live vaccines by cell surface engineering

通过细胞表面工程开发表位展示酵母作为活疫苗

基本信息

批准号：
12556012
负责人：
TANAKA Atsuo
金额：
$ 8.45万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12556012/
关键词：
Cell surface engineering Arming yeast Vaccine Epitope GPI-anchor Antigen Peptide Macrophage アーミング GPIアンカー抗原

项目摘要

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 … More 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

细胞表面是细胞内部和外部之间的功能界面。表面蛋白负责大多数细胞表面功能，充当细胞间粘附分子、特异性受体、酶和转运蛋白。细胞具有用于锚定表面特异性蛋白质并将表面蛋白质限制在细胞表面的特定结构域的系统。从应用的角度来看，应该利用已知的蛋白质向细胞表面的转运机制来开发细胞表面。建立这些在微生物细胞表面展示异源蛋白质的系统预计将有助于分离所产生的多肽以及微生物生物催化剂、全细胞吸附剂和活疫苗的生产。活细胞的细胞表面的利用对于微生物学和分子生物学的许多应用也很有吸引力。带有生物催化剂——葡糖淀粉酶、α-淀粉酶、CM-ce…更多纤维素酶、β-葡萄糖苷酶和脂肪酶——的新型酵母细胞是由酿酒酵母的细胞表面工程系统构建的。这些表面工程化的酵母细胞被称为“武装酵母”。编码米根霉葡糖淀粉酶及其分泌信号肽的基因与编码酵母α-凝集素C端一半的基因融合。通过细胞表面工程在细胞表面展示功能性蛋白质和肽，赋予酵母细胞新的功能。由于六组氨酸是众所周知的二价重金属离子螯合剂，因此尝试将该肽展示为酿酒酵母细胞表面的表位模型之一。这种细胞表面工程酵母吸附的铜离子比亲本菌株多三到八倍。通过 EDTA 处理，可以在不破碎细胞的情况下回收整个细胞吸附的大约一半的铜离子。此外，这种工程酵母将表现出铜离子耐受性，这表明酵母的新育种能够耐受有毒污染物。此外，为了定量展示在酵母细胞表面上的蛋白质和肽的量，构建了一种在其细胞表面上展示来自维多利亚水母的绿色荧光蛋白（GFP）的新型酵母菌株。这种细胞表面工程酵母菌株从细胞表面发出绿色荧光，可用于测量细胞表面上展示的分子数量，并通过各种启动子监测细胞内和/或细胞外条件的浓度。所利用的酿酒酵母菌株是表面工程酵母的第一个例子，其中靶向细胞表面的活性蛋白赋予细胞新的有益特性。较少的