The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Daniel A Fletcher - One of the best experts on this subject based on the ideXlab platform.
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molecular height measurement by Cell Surface optical profilometry csop
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Sungmin Son, Sho C Takatori, Brian Belardi, Marija Podolski, Matthew H Bakalar, Daniel A FletcherAbstract:The physical dimensions of proteins and glycans on Cell Surfaces can critically affect Cell function, for example, by preventing close contact between Cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native Cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of Cell Surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call Cell Surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model Cell, as well as to capture average protein and glycan heights on native Cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local Surface and solution conditions. We find that average height increases with Cell Surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native Cell Surface using antibodies and lectins. This versatile method for profiling Cell Surfaces has the potential to advance understanding of the molecular landscape of Cells and the role of the molecular landscape in Cell function.
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molecular height measurement by Cell Surface optical profilometry csop
bioRxiv, 2020Co-Authors: Sungmin Son, Sho C Takatori, Brian Belardi, Marija Podolski, Matthew H Bakalar, Daniel A FletcherAbstract:The physical dimensions of proteins and glycans on Cell Surfaces can critically affect Cell function, for example by preventing close contact between Cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native Cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of Cell Surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call Cell Surface optical profilometry (CSOP), to quantify height of key multi-domain proteins on a model macrophage and cancer Cell, as well as to capture average protein and glycan heights on native Cell membranes. We show that average height of a protein is significantly smaller than its contour length due to thermally driven bending and rotation on the membrane and that height strongly depends on local Surface and solution conditions. We find that average height increases with Cell Surface molecular crowding, while it decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native Cell Surface using antibodies and lectins. This versatile method for profiling Cell Surfaces has the potential to advance understanding of the molecular landscape of Cells and its role in Cell function.
Masahiro Zako - One of the best experts on this subject based on the ideXlab platform.
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functions of Cell Surface heparan sulfate proteoglycans
Annual Review of Biochemistry, 1999Co-Authors: Martin Götte, Pyong Woo Park, Ofer Reizes, Marilyn L Fitzgerald, John Lincecum, Masahiro ZakoAbstract:▪ Abstract The heparan sulfate on the Surface of all adherent Cells modulates the actions of a large number of extraCellular ligands. Members of both Cell Surface heparan sulfate proteoglycan families, the transmembrane syndecans and the glycosylphosphoinositide-linked glypicans, bind these ligands and enhance formation of their receptor-signaling complexes. These heparan sulfate proteoglycans also immobilize and regulate the turnover of ligands that act at the Cell Surface. The extraCellular domains of these proteoglycans can be shed from the Cell Surface, generating soluble heparan sulfate proteoglycans that can inhibit interactions at the Cell Surface. Recent analyses of genetic defects in Drosophila melanogaster, mice, and humans confirm most of these activities in vivo and identify additional processes that involve Cell Surface heparan sulfate proteoglycans. This chapter focuses on the mechanisms underlying these activities and on the Cellular functions that they regulate.
Akihiko Kondo - One of the best experts on this subject based on the ideXlab platform.
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mapping of endoglucanases displayed on yeast Cell Surface using atomic force microscopy
Colloids and Surfaces B: Biointerfaces, 2017Co-Authors: Musashi Takenaka, Chiaki Ogino, Takuya Kobayashi, Kentaro Inokuma, Tomohisa Hasunuma, Tatsuo Maruyama, Akihiko KondoAbstract:The Surface of yeast Cells has been an attractive interface for the effective use of Cellulose. Surface enzymes, however, are difficult to visualize and evaluate. In this study, two kinds of unique anchoring regions were used to display the Cellulase, endoglucanase (EG), on a yeast Cell Surface. Differences in the display level and the localization of EG were observed by atomic force microscopy. By surveying the yeast Cell Surface with a chemically modified cantilever, the interactive force between the Cellulose and EG was measured. Force curve mapping revealed differences in the display levels and the localization of EG according to anchoring regions. The proposed methodology enables visualization of displayed enzymes such as EG on the yeast Cell Surface.
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Cell Surface engineering of industrial microorganisms for biorefining applications
Biotechnology Advances, 2015Co-Authors: Tsutomu Tanaka, Akihiko KondoAbstract:In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignoCellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of Cell Surface display using industrial microorganisms such as Escherichia coli and yeast. Cell Surface display is used primarily for endowing Cellulolytic activity on the host Cells, and enables direct fermentation to generate useful fuels and chemicals from lignoCellulosic biomass. Cell Surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of Surface display for industrial biotechnology are discussed.
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Cell Surface display of enzymes by the yeast saccharomyces cerevisiae for synthetic biology
Fems Yeast Research, 2014Co-Authors: Tsutomu Tanaka, Akihiko KondoAbstract:In yeast Cell-Surface displays, functional proteins, such as Cellulases, are genetically fused to an anchor protein and expressed on the Cell Surface. Saccharomyces cerevisiae, which is often utilized as a Cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for Cell-Surface display. To construct yeast Cells with a desired function, such as the ability to utilize Cellulose as a substrate for bioethanol production, Cell-Surface display techniques for the efficient expression of enzymes on the Cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within Cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast Cell-Surface displays from a synthetic biology perspective and discuss approaches for further enhancing Cell-Surface display efficiency.
Martin Götte - One of the best experts on this subject based on the ideXlab platform.
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functions of Cell Surface heparan sulfate proteoglycans
Annual Review of Biochemistry, 1999Co-Authors: Martin Götte, Pyong Woo Park, Ofer Reizes, Marilyn L Fitzgerald, John Lincecum, Masahiro ZakoAbstract:▪ Abstract The heparan sulfate on the Surface of all adherent Cells modulates the actions of a large number of extraCellular ligands. Members of both Cell Surface heparan sulfate proteoglycan families, the transmembrane syndecans and the glycosylphosphoinositide-linked glypicans, bind these ligands and enhance formation of their receptor-signaling complexes. These heparan sulfate proteoglycans also immobilize and regulate the turnover of ligands that act at the Cell Surface. The extraCellular domains of these proteoglycans can be shed from the Cell Surface, generating soluble heparan sulfate proteoglycans that can inhibit interactions at the Cell Surface. Recent analyses of genetic defects in Drosophila melanogaster, mice, and humans confirm most of these activities in vivo and identify additional processes that involve Cell Surface heparan sulfate proteoglycans. This chapter focuses on the mechanisms underlying these activities and on the Cellular functions that they regulate.
Atsuo Tanaka - One of the best experts on this subject based on the ideXlab platform.
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spacer mediated display of active lipase on the yeast Cell Surface
Applied Microbiology and Biotechnology, 2001Co-Authors: Motohisa Washida, Mitsuyoshi Ueda, Satomi Takahashi, Atsuo TanakaAbstract:We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the Cell Surface by Cell Surface engineering. The gene encoding Rhizopus oryzae lipase (ROL) was fused with the genes encoding the pre-α-factor leader sequence and the C-terminal half of α-agglutinin including the glycosylphosphatidylinositol-anchor attachment signal. The constructed gene was overexpressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance lipase activity by preserving the conformation of the active site near the C-terminal portion. Localization of the expressed ROL on the Cell Surface was confirmed by immunofluorescence microscopy. The ROL displayed on the yeast Cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1-ol tributyl ester (BALB) and insoluble triolein. The insertion of linker peptides effected the activity towards BALB, thereby demonstrating that the optimal length of linker peptides was present. The activity towards triolein was higher in lipases with longer linker peptides. ROL displayed on the Cell wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme. This is the first report of an active lipase displayed on the Cell Surface. Furthermore, insertion of a linker peptide of the appropriate length as a spacer may be an improved method to effectively display enzymes, especially those having the active region at the C-terminal portion, on the Cell Surface.
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genetic immobilization of proteins on the yeast Cell Surface
Biotechnology Advances, 2000Co-Authors: Mitsuyoshi Ueda, Atsuo TanakaAbstract:Abstract A genetic system has been exploited to immobilize proteins in their active and functional forms on the Cell Surface of yeast, Saccharomyces cerevisiae. DNAs encoding proteins with a secretion signal peptide were fused with the genes encoding yeast agglutinins, a- and α-type proteins involved in mating. The fusion gene was introduced into S. cerevisiae and expressed under the control of several promoters. Appearance of the fused proteins expressed on the Cell Surface was demonstrated biochemically and by immunofluorescence and immunoelectron microscopy techniques. α-Galactosidase from Cyamopsis tetragonoloba seeds, peptide libraries including scFv and variable regions of the T Cell receptor from mammalian Cells have been successfully immobilized on the yeast Cell wall in the active form. Recently, Surface-engineered yeasts have been constructed by immobilizing the enzymes and a functional protein, for example, green fluorescent protein (GFP) from Aequorea victoria. The yeasts were termed ‘arming yeasts’ with biocatalysts or functional proteins. Such arming Cells displaying glucoamylase from Rhizopus oryzae and α-amylase from Bacillus stearothermophilus , or carboxymethylCellulase and β-glucosidase from Aspergillus acleatus , could assimilate starch or Cellooligosaccharides as the sole carbon source, although S. cerevisiae cannot intrinsically assimilate these substrates. GFP-arming Cells can emit green fluorescence from the Cell Surface in response to the environmental conditions. The approach described in this review will enable us to endow living Cells, including yeast Cells, with novel additional abilities and to open new dimensions in the field of biotechnology.
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Cell Surface engineering of yeast construction of arming yeast with biocatalyst
Journal of Bioscience and Bioengineering, 2000Co-Authors: Mitsuyoshi Ueda, Atsuo TanakaAbstract:A Cell Surface engineering system of yeast Saccharomyces cerevisiae has been established and novel yeasts armed by biocatalysts (enzymes-glucoamylase, alpha-amylase, CM-Cellulase, beta-glucosidase, and lipase), termed "arming yeasts", were constructed. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half of yeast alpha-agglutinin and expressed in S. cerevisiae. Glucoamylase was shown to be displayed on the Cell Surface in its active form and anchored covalently to the Cell wall. S. cerevisiae itself is unable to utilize starch, while the Surface-engineered yeast could grow on starch as the sole carbon source. For further improvement of the ability to directly ferment starchy materials by the Cell Surface-engineered yeast, engineered yeasts displaying two amylolytic enzymes on the Cell Surface were constructed. The gene encoding R. oryzae glucoamylase with its own secretion signal peptide and a truncated fragment of the alpha-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast alpha-factor were fused with the gene encoding the C-terminal half of the yeast alpha-agglutinin. The Surface-engineered yeast co-displaying glucoamylase and alpha-amylase by the integration of their genes into the chromosomes could grow faster on starch as the sole carbon source than the engineered Cells displaying only glucoamylase. The system was further applied to the construction of a novel Cellulose-utilizing yeast by displaying Cellulolytic enzymes in their active form on the Cell Surface of S. cerevisiae. Engineered yeasts co-displaying FI-carboxymethylCellulase (CM-Cellulase), one of the endo-type Cellulases, and beta-glucosidase from Aspergillus aculeatus on their Cell Surface were also constructed. The yeasts displaying these Cellulases were given the ability to assimilate Cellooligosaccharide, suggesting the possibility that the assimilation of Cellulosic materials may be carried out by S. cerevisiae displaying heterologous Cellulase proteins on the Cell Surface. The system has also been used for the Cell Surface display of R. oryzae lipase (ROL). Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance the lipase activity. The insertion of an appropriate length of a linker peptide as a spacer is effective in the display of ROL, having the active region at the C-terminal portion, on the Cell Surface. Thus, Cell Surface engineering will be capable of conferring novel additional abilities upon living Cells and will herald a new era in the field of biotechnology.