Yeast
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Cell wall structure suitable for surface display of proteins in <i>Saccharomyces cerevisiae</i> Abstract A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae , the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β ‐Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9‐fold higher than that of the wild‐type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild‐type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild‐type strain. The relative value (mnn2 deletion mutant/wild‐type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β ‐glucosidase activity using p ‐nitrophenyl‐β ‐glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high‐molecular‐weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.
Yeast - Tập 31 Số 2 - Trang 67-76 - 2014
Three yeast genes, <i>PIR1</i>, <i>PIR2</i> and <i>PIR3</i>, containing internal tandem repeats, are related to each other, and <i>PIR1</i> and <i>PIR2</i> are required for tolerance to heat shock Abstract We isolated three highly homologous genes, PIR1, PIR2 and PIR3 , collectively called the PIR genes. The remarkable feature of their putative amino acid sequence is that they contain a sequence consisting of 18–19 amino acid residues repeated tandemly seven to ten times. Genes homologous to PIR were found in Kluyveromyces lactis and Zygosaccharomyces rouxii but not in Schizosaccharomyces pombe , suggesting that a set of PIR genes plays some role in budding yeast. Bias of codon usage seen in each of the PIR translation products suggests that they are expressed abundantly. The fact that disruption of each gene is viable indicates that none of them is essential. The double disruptants, pir1 pir2 , were viable under various conditions, such as higher temperature (37°C) or high salt concentration, but showed a slow‐growing phenotype on an agar slab. Furthermore, they were sensitive to heat shock. Addition of a pir3 disruption to the pir1 pir2 double disruptant brought about no phenotypic difference from the original double mutant. PIR1 and PIR3 are closely linked to each other and are on chromosome XI.
Yeast - Tập 9 Số 5 - Trang 481-494 - 1993
Isolation of <i>Pichia pastoris PIR</i> genes and their utilization for cell surface display and recombinant protein secretion Abstract Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two p roteins with i nternal r epeats (PIR ) genes, i.e. PpPIR1 and PpPIR2 , from the methylotrophic yeast Pichia pastoris has been reported. The PIR1 and PIR2 genes' open reading frames were found to contain 1068 and 972 bases, respectively. The sequence homology search showed a homologous conserved repeat of PIR yeast block (SQIGDGQIQATT) in both proteins. The PIR yeast block was present eight times in the PpPir1p and four times in the PpPir2p proteins. Both proteins showed conserved glutamine (Q) and aspartic acid (D) in the repeated sequences, indicating a possible alkali‐sensitive β1,3‐glucan ester linkage. The fusion constructs of PpPir1‐2p and enhanced green fluorescent protein (EGFP) were developed for yeast cell surface display. The immunofluorescence assay showed uniform localization of EGFP protein on the P. pastoris cell surface in all fusion constructs. Furthermore, new vectors were developed for recombinant protein secretion in P. pastoris , utilizing the pre‐pro signal of PpPir1p protein. Efficient processing of the signal sequence was observed from EGFP and human α1‐antitrypsin (AAT) fusion constructs and recombinant protein secretion was obtained in the culture supernatant. The DNA sequences of the P. pastoris PpPIR1 and PpPIR2 genes have been submitted to GenBank under Accession Nos HM446634 and HM446635, respectively. Copyright © 2010 John Wiley & Sons, Ltd.
Yeast - Tập 28 Số 3 - Trang 213-226 - 2011
A cell surface display system using novel GPI‐anchored proteins in <i>Hansenula polymorpha</i> Abstract A cell surface display system was developed in yeast Hansenula polymorpha. The four genes HpSED1 , HpGAS1 , HpTIP1 and HpCWP1 , encoding glycosylphosphatidyl‐inositol (GPI)‐anchored cell surface proteins from H. polymorpha , were cloned, characterized and evaluated for their efficacies as cell surface display motifs of reporter proteins. Sequence analysis of these genes revealed that each encodes a typical GPI‐anchored protein that is structurally similar to a counterpart gene in S. cerevisiae . The genes showed a high content of serine‐threonine (alanine) and harboured a putative secretion signal in the N‐terminus and the GPI‐attachment signal in the C‐terminus. The surface anchoring efficiency of these putative cell surface proteins was tested by fusion to the C‐terminal of carboxymethylcellulase (CMCase) from Bacillus subtilis . In all cases, high CMCase activities were detected in intact cell fraction, indicating anchoring of CMCase to the cell surface. HpCwp1p, HpGas1p and the 40 C‐terminal amino acids of HpTip1p from H. polymorpha exhibited a comparatively high CMCase surface anchoring efficiency. When these proteins were used as anchoring motifs for surface display of the glucose oxidase (GOD) from Aspergillus niger , most enzyme activity was detected at the cell surface. Fluorescence activated cell sorter (FACS) analysis of cells displaying GOD on the cell surface demonstrated that GOD was well exposed on the cell surface. HpCwp1p showed the highest anchoring efficiency among others. Copyright © 2002 John Wiley & Sons, Ltd.
Yeast - Tập 19 Số 13 - Trang 1153-1163 - 2002
Genome‐wide identification of fungal GPI proteins Abstract Glycosylphosphatidylinositol‐modified (GPI) proteins share structural features that allow their identification using a genomic approach. From the known S. cerevisiae and C. albicans GPI proteins, the following consensus sequence for the GPI attachment site and its downstream region was derived: [NSGDAC]–[GASVIETKDLF]–[GASV]–X(4,19)–[FILMVAGPSTCYWN](10)>, where > indicates the C‐terminal end of the protein. This consensus sequence, which recognized known GPI proteins from various fungi, was used to screen the genomes of the yeasts S. cerevisiae , C. albicans , Sz. pombe and the filamentous fungus N. crassa for putative GPI proteins. The subsets of proteins so obtained were further screened for the presence of an N‐terminal signal sequence for the secretion and absence of internal transmembrane domains. In this way, we identified 66 putative GPI proteins in S. cerevisiae . Some of these are known GPI proteins that were not identified by earlier genomic analyses, indicating that this selection procedure renders a more complete image of the S. cerevisiae GPI proteome. Using the same approach, 104 putative GPI proteins were identified in the human pathogen C. albicans . Among these were the proteins Gas/Phr, Ecm33, Crh and Plb, all members of GPI protein families that are also present in S. cerevisiae . In addition, several proteins and protein families with no significant homology to S. cerevisiae proteins were identified, including the cell wall‐associated Als, Csa1/Rbt5, Hwp1/Rbt1 and Hyr1 protein families. In Sz. pombe , which has a low level of (galacto)mannan in the cell wall compared to C. albicans and S. cerevisiae , only 33 GPI candidates were identified and in N. crassa 97. BLAST searches revealed that about half of the putative GPI proteins that were identified in Sz. pombe and N. crassa are homologous to known or putative GPI proteins from other fungi. We conclude that our algorithm is selective and can also be used for GPI protein identification in other fungi. Copyright © 2003 John Wiley & Sons, Ltd.
Yeast - Tập 20 Số 9 - Trang 781-796 - 2003
Functional analysis of the cysteine residues and the repetitive sequence of <i>Saccharomyces cerevisiae</i> Pir4/Cis3: the repetitive sequence is needed for binding to the cell wall β‐1,3‐glucan Abstract Identification of PIR/CIS3 gene was carried out by amino‐terminal sequencing of a protein band released by β‐mercaptoethanol (β‐ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with β‐1,3‐glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys214 ‐12aa‐Cys227 ‐COOH), reduced but did not eliminate incorporation of Pir4 (p rotein with i nternal r epeats) by disulphide bridges. Similarly, site‐directed mutation of two other cysteine amino acids (Cys130 Ser or Cys197 Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2‐unprocessed Pir4. Therefore, it seems that deletion or mutation of individual cysteine molecules does not seem enough to inhibit incorporation of Pir4 by disulphide bridges. In fks1Δ and gsc2/fks2Δ cells, defective in β‐1,3‐glucan synthesis, modification of the protein pattern found in the supernatant of the growth medium, as well as the material released by β‐ME or laminarinase, was evident. However, incorporation of Pir4 by both disulphide bridges and to the β‐1,3‐glucan of the cell wall continued. Deletion of the repetitive sequence (QIGDGQVQA) resulted in the secretion and incorporation by disulphide bridges of Pir4 in reduced amounts together with substantial quantities of the Kex2‐unprocessed Pir4 form. Pir4 failed to be incorporated in alkali‐sensitive linkages involving β‐1,3‐glucan when the first repetitive sequence was deleted. Therefore, this suggests that this sequence is needed in binding Pir4 to the β‐1,3‐glucan. Copyright © 2003 John Wiley & Sons, Ltd.
Yeast - Tập 20 Số 11 - Trang 973-983 - 2003
Targeting of a heterologous protein to the cell wall of <i>Saccharomyces cerevisiae</i> Abstract The sexual adhesion protein of Saccharomyces cerevisiae MATα cells, α‐agglutinin, could not be extracted from the cell wall with hot sodium dodecyl sulfate (SDS), but became soluble after digestion of the cell with laminarinase. This indicates that it is intimately associated with cell wall glucan. A fusion protein was constructed consisting of the signal sequence of yeast invertase, guar α‐galactosidase, and the C‐terminal half of the α‐agglutinin. Most of the fusion protein was incorporated in the cell wall. A small amount could be extracted with SDS, but most of it could only be extracted with laminarinase. On the other hand, cells containing a construct consisting of the signal sequence of invertase and α‐galactosidase released most of the α‐galactosidase into the medium and all cell wall‐associated α‐galactosidase was released by SDS. Labelling with antibodies showed that the α‐galactosidase part of the fusion protein was exposed on the surface of the cell wall. The results demonstrate that the C‐terminal half of the α‐agglutinin contains the information needed to incorporate a protein into the cell wall.
Yeast - Tập 9 Số 4 - Trang 399-409 - 1993
Deletion of <i>PDE2</i>, the gene encoding the high‐affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in <i>Candida albicans</i> Abstract A role for the cAMP‐dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2 , the gene encoding the high cAMP‐affinity phosphodiesterase. Unlike wild‐type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild‐type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60–65 nm, compared to 80–85 nm in the wild‐type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans . Copyright © 2005 John Wiley & Sons, Ltd.
Yeast - Tập 22 Số 4 - Trang 285-294 - 2005
Sequence, mapping and disruption of <i>CCC2</i>, a gene that cross‐complements the Ca<sup>2+</sup>‐sensitive phenotype of <i>csg1</i> mutants and encodes a P‐type ATPase belonging to the Cu<sup>2+</sup>‐ATPase subfamily Abstract We have isolated, sequenced, mapped and disrupted a gene, CCC2 , from Saccharomyces cerevisiae . This gene displays non‐allelic complementation of the Ca2+ ‐sensitive phenotype conferred by the csg1 mutation. Analysis of the CCC2p amino acid sequence reveals that it encodes a member of the P‐type ATPase family and is most similar to a subfamily thought to consist of Cu2+ transporters, including the human genes that mutate to cause Wilson disease and Menkes disease. The ability of this gene, in two or more copies, to reverse the csg1 defect suggests that Ca2+ ‐induced death of csg1 mutant cells is related to Cu2+ metabolism. Cells without CCC2 require increased Cu2+ concentrations for growth. Therefore CCC2p may function to provide Cu2+ to a cellular compartment rather than in removal of excess of Cu2+ . The sequence of CCC2 is available through GenBank under accession number L36317.
Yeast - Tập 11 Số 3 - Trang 283-292 - 1995
Yeast/<i>E. coli</i> shuttle vectors with multiple unique restriction sites Abstract Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomuosly in Saccharomyces cerevisiae and in E. coli; (2) they contain the β‐lactamase gene and confer ampicillin resistance to E. coli ; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI , SacI , KpnI , SmaI , BamH1 , XbaI , SbaI , SalI , PstI , SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI , which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of β‐galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these grown under non‐selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for constructions of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for antunomous replication in yeast. The derivative plasmids YIp651 and YIp352 can therefore be used ti integrate specific sequences into yeast chromosomal DNA.
Yeast - Tập 2 Số 3 - Trang 163-167 - 1986
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