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The cell surface display system in yeast enables the innovative strategy for improving cellular functions in a wide range of applications such as biofuel production, bioremediation, synthesis of valuable chemicals, recovery of rare metal ions, development of biosensors, and high-throughput screening of proteins/peptides library. Display of enzymes for polysaccharide degradation enables the construction of metabolically engineered whole-cell biocatalyst owing to the accessibility of the displayed enzymes to high-molecular-weight polysaccharides. In addition, along with fluorescence-based activity evaluation, fluorescence-activated cell sorting (FACS), and yeast cell chip, the cell surface display system is an effective molecular tool for high-throughput screening of mutated proteins/peptides library. In this article, we describe the methods for cell surface display of proteins/peptides of interest on yeast, evaluation of display efficiency, and harvesting of the displayed proteins/peptides from cell surface.

Convenient detection of Candida albicans in human bodies is essential to treat patients with candidiasis. In this study, we designed a fluorescence-quenched peptide that emits fluorescence after being cleaved by virulent proteases secreted by C. albicans. The designed peptide will enable easy detection of candidiasis in immunocompromised patients., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The budding yeast, Saccharomyces cerevisiae, is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae, as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae.

GTS1 of Saccharomyces cerevisiae is a pleiotropic gene. Its induction leads to a variety of biological phenomena represented by cell aggregation. The C-terminal polyglutamine sequence in Gts1p is indispensable for its pleiotropy and nuclear localization. This sequence is often observed in polyglutamine diseases, such as Huntington disease, and is believed to induce protein aggregation, leading to cell death. In this study, protein aggregates were formed in a polyglutamine-dependent manner in cells inducing GTS1, and heat-shock protein family, translation elongation factor, and mitochondrial proteins were trapped in Gts1p-mediated protein aggregates. Moreover, the polyglutamine sequence of Gts1p was indispensable to the induction of reactive oxygen species (ROS) production and apoptosis. Deletion of the genes encoding Por1p and Yhb1p altered the profiles of ROS production and apoptosis caused by GTS1 induction, suggesting that the trapping of these proteins in Gts1p-mediated protein aggregates inhibits the intrinsic functions of these proteins.

The cell wall of Saccharomyces cerevisiae plays an essential role in the biophysical characteristics of the cell surface. The modification of the cell wall property is an important factor for cellular adaptation to a stressful environment. In this study, we randomly modified the cell wall by displaying combinatorial random peptides on the yeast cell surface, and by screening, we successfully obtained a novel peptide, Scr35, that endowed yeasts with acid tolerance. The yeast, surface-modified by Scr35, was able to grow well under acidic condition and low glucose condition and showed high glucose uptake activity. However, the growth of the modified yeast became inferior as extracellular pH became higher. This inferiority was rescued by decreasing glucose concentration in a medium. Our results suggest that the optimum pH of a medium becomes low when the newly created Scr35 affects glucose uptake activity through cell-surface modification. Therefore, such artificial modification of the cell surface has a great potential as a useful tool for breeding acid-tolerant yeasts for industrial applications of S. cerevisiae as a biocatalyst.

Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity. [ABSTRACT FROM AUTHOR]

Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called "arming technology", can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach. [ABSTRACT FROM AUTHOR]

The article presents the findings of a research conducted on the identification of interaction site of propeptide toward mature carboxypeptidase Y (mCPY), based on the similarity between propeptide and CPY inhibitor (IC). Under the research, sequence alignment between IC and proCPY indicated that a sequence similar to the N-terminal region of IC was present in proCPY. It concludes that C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.

The article presents the findings of a research conducted on the identification of interaction site of propeptide toward mature carboxypeptidase Y (mCPY), based on the similarity between propeptide and CPY inhibitor (IC). Under the research, sequence alignment between IC and proCPY indicated that a sequence similar to the N-terminal region of IC was present in proCPY. It concludes that C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.

The article presents the findings of a research conducted on the identification of interaction site of propeptide toward mature carboxypeptidase Y (mCPY), based on the similarity between propeptide and CPY inhibitor (IC). Under the research, sequence alignment between IC and proCPY indicated that a sequence similar to the N-terminal region of IC was present in proCPY. It concludes that C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.

The genetic engineering of microorganisms to adsorb metal ions is an attractive method to facilitate the environmental cleanup of metal pollution and to enrich the recovery of metal ions such as rare metal ions. For the recovery of metal ions by microorganisms, cell surface design is an effective strategy for the molecular breeding of bioadsorbents as an alternative to intracellular accumulation. The cell surface display of known metal-binding proteins/peptides and the molecular design of novel metal-binding proteins/peptides have been performed using a cell surface engineering approach. The adsorption of specific metal ions is the important challenge for the practical recovery of metal ions. In this paper, we discuss the recent progress in surface-engineered bioadsorbents for the recovery of metal ions. [Copyright &y& Elsevier]