Yeast-enhanced Green Fluorescent Protein Research Articles

Engineering of the hypoxia-induced Pichia stipitis ADH2 promoter to construct a promoter library for Pichia pastoris.

Hypoxia-inducible promoters of a wide range of activities are desirable for fine-tuning gene expression in response to oxygen limitation, especially for the Crabtree negative yeast Pichia pastoris (Komagataella phaffii) with a high oxygen consumption rate in large-scale fermentations. Here we constructed a hypoxia-inducible promoter library for P. pastoris through error-prone PCR of Pichia stipitis ADH2 promoter (PsADH2). The library of 30 selected promoters showing 0.4- to 5.5-fold of the PsADH2 activity was obtained through high-throughput screening in microplates using the reporter yeast-enhanced green fluorescent protein. Two strong promoters, AM23 and AM30, were further characterized in shake flask cultures at high and low dissolved oxygen levels. They responded more sensitively to the low dissolved oxygen level, achieving a 4.6-, 7.9-fold and 3.6-, 7.7-fold higher fluorescence intensity and transcript level, respectively, than the wild-type PsADH2. Their hypoxia-inducible properties were confirmed with two additional reporters: β-galactosidase and Vitreoscilla hemoglobin, to demonstrate the broad applicability of the promoter library. During the typical fermentation process in shake flasks, the promoter AM30 showed strong expression with cell growth and decreased oxygen levels, without any additional chemical inducers or operations. Since the potent industrial host P. pastoris is recognized as an easy to scale-up system, it is reasonable to expect that the obtained hypoxia-inducible promoter library may have great potential to enable convenient regulation of gene expression under industrial fermentations which are usually run under oxygen limitation due to high cell density cultivations.

AOX1 promoter-driven expression of yeast-enhanced green fluorescent protein in Pichia pastoris

Green fluorescent protein (GFP) is a widely employed reporter for studying various cellular functions in genetic engineering. Methylotrophic yeast; Pichia pastoris is an excellent host for heterologous protein production. Codon optimized yeast-enhanced green fluorescent protein (yEGFP) has been reported to have higher expression as a yeast reporter which can be used to study many aspects of P. pastoris. The present investigation was focused on studying the yEGFP intracellular expression pattern in the P. pastoris GS115 strain. yEGFP is cloned into the pPICZ A vector under the control of an AOX1 (Alcohol Oxidase) promoter that was induced by adding methanol to the growth medium. However, methanol concentration and time of induction could affect the optimum expression of yEGFP. Hence, those conditions were tested for manifesting optimum yEGFP expression by monitoring the fluorescence intensities in minimal methanol histidine media supplemented with 0.5%, 1%, 2%, and 2.5% methanol. The pH of the media and the composition of the media (minimal or complex) also affect recombinant protein production. The effect of pH and the media composition was investigated in buffered media at pH 6 and unbuffered media of minimal methanol histidine media and methanol complex media. The results showed that the yEGFP expression increased up to the 5th day of induction and then decreased on the 6th day, 2% methanol showed the highest expression, and the pH and the media composition did not have any effect on the yEGFP expression. In conclusion, the optimum yEGFP expression can be obtained in 2% methanol on the 5th day of induction irrespective of minimal or complex media used.

Development of surface displaying system for heterologous protein expression in Candida tropicalis

The purpose of this study was to assess the potential application of cell surface display in Candida tropicalis. Surface display gene cassettes were constructed using five anchoring proteins from Saccharomyces cerevisiae, three of which [(suppression of exponential defect protein, SED1), (cell wall protein 2, CWP2) and (delayed anaerobic protein 4, DAN4)] were reported to show higher activity of heterologous proteins than α-agglutinin (AGα1). The performance of yeast-enhanced green fluorescent protein (yeGFP) was evaluated using laser scanning confocal microscopy and flow cytometry. The results showed that the three anchoring regions (SED1, CWP2 and AGα1) successfully displayed yeGFP on the cell wall. To investigate the effect of the three anchoring proteins on the surface display of Rhizopus oryzae α-amylase (ROA1) and Aspergillus aculeatus β-glucosidase (BGL1) in C. tropicalis, we constructed surface display gene cassettes for ROA1 and BGL1, respectively. The strains containing the anchoring proteins SED1 and CWP2 showed higher activity of ROA1 and BGL1 than the strains containing the anchoring protein AGα1. The highest ROA1 and BGL1 activities of strains with SED1 were 6.37 U/g CDW and 7.93 U/g CDW, respectively, which were sixfold and eightfold higher than those of strain with AGα1. In addition, we also optimized signal peptides. The results indicated that signal peptides have an impact on enzyme activity.

Screening endogenous signal peptides and protein folding factors to promote the secretory expression of heterologous proteins in Pichia pastoris

Secretory expression is most often desired but usually hampered by limitations of signal peptide processing and protein folding in the methylotrophic yeast Pichia pastoris. To alleviate such limitations, novel endogenous signal peptides (Dan4, Gas1, Msb2, and Fre2) and folding factors (Mpd1p, Pdi2p, and Sil1p) were predicted based on the reported P. pastoris secretome and genome. Their effects were investigated using three reporter proteins: yeast-enhanced green fluorescent protein (yEGFP), β-galactosidase (Gal) and cephalosporin C acylase (SECA), in comparison with the commonly used Saccharomyces cerevisiae alpha-mating factor pre-pro leader sequence (α-MF) or folding factors (Pdi1p, BiP, and Hac1p). The newly identified signal sequences were superior over α-MF for production of heterologous proteins. The signal peptide Msb2 increased the specific extracellular production of all reporter proteins, ranging from 1.5- to 8.0-fold, and Dan4 enhanced all total protein production up to 172-fold. Co-expression of folding factors exhibited a protein-specific effect on cell growth, transcription and expression of different reporter genes. All of the novel folding factors enhanced total production of SECA, and Sil1p performed best in the extracellular SECA production, showing a 3.3-fold increase. These novel signal peptides and folding factors can be used for promoting secretion of heterologous proteins in P. pastoris.

Alcohol Acetyltransferase Eat1 Is Located in Yeast Mitochondria.

Eat1 is a recently discovered alcohol acetyltransferase responsible for bulk ethyl acetate production in yeasts such as Wickerhamomyces anomalus and Kluyveromyces lactis These yeasts have the potential to become efficient bio-based ethyl acetate producers. However, some fundamental features of Eat1 are still not understood, which hampers the rational engineering of efficient production strains. The cellular location of Eat1 in yeast is one of these features. To reveal its location, Eat1 was fused with yeast-enhanced green fluorescent protein (yEGFP) to allow intracellular tracking. Despite the current assumption that bulk ethyl acetate production occurs in the yeast cytosol, most of Eat1 localized to the mitochondria of Kluyveromyces lactis CBS 2359 Δku80 We then compared five bulk ethyl acetate-producing yeasts in iron-limited chemostats with glucose as the carbon source. All yeasts produced ethyl acetate under these conditions. This strongly suggests that the mechanism and location of bulk ethyl acetate synthesis are similar in these yeast strains. Furthermore, an in silico analysis showed that Eat1 proteins from various yeasts were mostly predicted as mitochondrial. Altogether, it is concluded that Eat1-catalyzed ethyl acetate production occurs in yeast mitochondria. This study has added new insights into bulk ethyl acetate synthesis in yeast, which is relevant for developing efficient production strains.IMPORTANCE Ethyl acetate is a common bulk chemical that is currently produced from petrochemical sources. Several Eat1-containing yeast strains naturally produce large amounts of ethyl acetate and are potential cell factories for the production of bio-based ethyl acetate. Rational design of the underlying metabolic pathways may result in improved production strains, but it requires fundamental knowledge on the function of Eat1. A key feature is the location of Eat1 in the yeast cell. The precursors for ethyl acetate synthesis can be produced in multiple cellular compartments through different metabolic pathways. The location of Eat1 determines the relevance of each pathway, which will provide future targets for the metabolic engineering of bulk ethyl acetate production in yeast.

Natural yeast promoter variants reveal epistasis in the generation of transcriptional-mediated noise and its potential benefit in stressful conditions.

The increase in phenotypic variability through gene expression noise is proposed to be an evolutionary strategy in selective environments. Differences in promoter-mediated noise between Saccharomyces cerevisiae strains could have been selected for thanks to the benefit conferred by gene expression heterogeneity in the stressful conditions, for instance, those experienced by industrial strains. Here, we used a genome-wide approach to identify promoters conferring high noise levels in the industrial wine strain EC1118. Many promoters of genes related to environmental factors were identified, some of them containing genetic variations compared with their counterpart in the laboratory strain S288c. Each variant of eight promoters has been fused to yeast-Enhanced Green Fluorescent Protein and integrated in the genome of both strains. Some industrial variants conferred higher expression associated, as expected, with lower noise, but other variants either increased or decreased expression without modifying variability, so that they might exhibit different levels of transcriptional-mediated noise at equal mean. At different induction conditions giving similar expression for both variants of the CUP1 promoter, we indeed observed higher noise with the industrial variant. Nevertheless, this difference was only observed in the industrial strain, revealing epistasis in the generation of promoter-mediated noise. Moreover, the increased expression variability conferred by this natural yeast promoter variant provided a clear benefit in the face of an environmental stress. Thus, modulation of gene expression noise by a combination of promoter modifications and trans-influences might be a possible adaptation mechanism in yeast.

An enhanced approach for engineering thermally stable proteins using yeast display

Many biotechnology applications require the evolution of enhanced protein stability. Using polymerase chain reaction-based recovery of engineered clones during the screen enrichment phase, we describe a yeast display method capable of yielding engineered proteins having thermal stability that substantially exceeds the viability threshold of the yeast host. To this end, yeast-enhanced green fluorescent protein destabilized by dual-loop insertion was engineered to possess a substantially enhanced resistance to thermal denaturation at 70°C. Stabilized proteins were secreted, purified and found to have three- to six-fold increased resistance to thermal denaturation. The validated method enables yeast display-based screens in previously inaccessible regions of the fitness landscape.

Agonistic and antagonistic estrogens in licorice root (Glycyrrhiza glabra).

The roots of licorice (Glycyrrhiza glabra) are a rich source of flavonoids, in particular, prenylated flavonoids, such as the isoflavan glabridin and the isoflavene glabrene. Fractionation of an ethyl acetate extract from licorice root by centrifugal partitioning chromatography yielded 51 fractions, which were characterized by liquid chromatography–mass spectrometry and screened for activity in yeast estrogen bioassays. One third of the fractions displayed estrogenic activity towards either one or both estrogen receptors (ERs; ERα and ERβ). Glabrene-rich fractions displayed an estrogenic response, predominantly to the ERα. Surprisingly, glabridin did not exert agonistic activity to both ER subtypes. Several fractions displayed higher responses than the maximum response obtained with the reference compound, the natural hormone 17β-estradiol (E2). The estrogenic activities of all fractions, including this so-called superinduction, were clearly ER-mediated, as the estrogenic response was inhibited by 20–60% by known ER antagonists, and no activity was found in yeast cells that did not express the ERα or ERβ subtype. Prolonged exposure of the yeast to the estrogenic fractions that showed superinduction did, contrary to E2, not result in a decrease of the fluorescent response. Therefore, the superinduction was most likely the result of stabilization of the ER, yeast-enhanced green fluorescent protein, or a combination of both. Most fractions displaying superinduction were rich in flavonoids with single prenylation. Glabridin displayed ERα-selective antagonism, similar to the ERα-selective antagonist RU 58668. Whereas glabridin was able to reduce the estrogenic response of E2 by approximately 80% at 6 × 10−6 M, glabrene-rich fractions only exhibited agonistic responses, preferentially on ERα. Figure Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-5061-9) contains supplementary material, which is available to authorized users.

Reliable high-throughput approach for screening of engineered constitutive promoters in the yeast Pichia pastoris

To develop a reliable and sensitive high-throughput approach for the screening of engineered constitutive promoters in the yeast Pichia pastoris. The yeast-enhanced green fluorescent protein (yEGFP) was used as the reporter to monitor the promoter strength. After eliminating the interfering components (yeast extract and tryptone) with fluorescence signal from the medium, a high-throughput screening approach was established and optimized to obtain a low standard deviation of cell density (6.9%) and fluorescence (7.4%) in 48-deep-well microplates. Then, 300 clones containing GAP promoter (P(GAP)) variants were screened, exhibiting a wide range in fluorescent intensity from about 8% to 218% of that obtained with P(GAP). Six representative clones with unique promoter sequence were picked for further characterization. A good correlation between yEGFP fluorescence in microplates and shake flasks was observed. Furthermore, the high correlation between fluorescence and transcript levels confirmed that expression was transcriptionally controlled. We developed a reliable high-throughput screening approach that can be used to select engineered constitutive promoters of varying strengths. This approach is expected to accelerate the selection of constitutive promoters in P. pastoris and can also be applied for the screening of other constitutive expression clones.

GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.

Gynaecomastia linked to the intake of a herbal supplement fortified with diethylstilbestrol

This study reports the findings of a supplement marketed on the Internet for prostate problems. The supplement was orally taken by a 60-year-old man with divergent hormonal levels and who was surgically treated for gynaecomastia: development of abnormally large mammary glands in males. The supplement showed a strong effect in a yeast oestrogen bioassay, expressing a yeast-enhanced green fluorescent protein (yEGFP) upon exposure to oestrogens. Using both nuclear magnetic resonance (NMR) and a gradient liquid chromatographic time-of-flight mass spectrometric (LC/TOF-MS) method, the response was shown to be caused by very high levels of diethylstilbestrol, known for causing gynaecomastia. The gynaecomastia was most probably caused by this orally taken ‘natural’ herbal supplement, as the patient's hormonal levels also returned to normal again when stopping the use of it. This case demonstrates that physicians need to be aware of the use of supplements with illegal components that may be responsible for unwanted side-effects.

Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro

Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a K(d) of approximately 82 nm. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4-8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosaccharides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was autophosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.

A surface display yeast two-hybrid screening system for high-throughput protein interactome mapping

Despite the wide acceptance of yeast two-hybrid (Y2H) system for protein–protein interaction analysis and discovery, conventional Y2H assays are not well suited for high-throughput screening of the protein interaction network (“interactome”) on a genomic scale due to several limitations, including labor-intensive agar plating and colony selection methods associated with the use of nutrient selection markers, complicated reporter analysis methods associated with the use of LacZ enzyme reporters, and incompatibility of the liquid handling robots. We recently reported a robust liquid culture Y2H system based on quantitative analysis of yeast-enhanced green fluorescent protein (yEGFP) reporters that greatly increased the analysis throughput and compatibility with liquid handling robots. To further advance its utility in high-throughput complementary DNA (cDNA) library screening, we report the development of a novel surface display Y2H (sdY2H) library screening system with uniquely integrated surface display hemagglutination (sdHA) antigen and yEGFP reporters. By introduction of a surface reporter sdHA into the yEGFP-based Y2H system, positive Y2H targets are quickly isolated from library cells by a simple magnetic separation without a large plating effort. Moreover, the simultaneous scoring of multiple reporters, including sdHA, yEGFP, and conventional nutrient markers, greatly increased the specificity of the Y2H assay. The feasibility of the sdY2H assay on large cDNA library screening was demonstrated by the successful recovery of positive P53/T interaction pairs at a target-to-background ratio of 1:1,000,000. Together with the massive parallel DNA sequencing technology, it may provide a powerful proteomic tool for high-throughput interactome mapping on a genomic scale.

Secretion and Surface Display of Green Fluorescent Protein Using the Yeast Saccharomyces cerevisiae

Green fluorescent protein (GFP) continues to be a very useful tool in biotechnology, but soluble production of GFP and GFP-protein fusions has been difficult. In this study, we have produced yeast-enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a soluble, secreted product with a purified level of 6 mg/L. Expression was directed by the inducible GAL1-10 promoter and synthetic prepro leader sequence. The secretion of yEGFP by yeast was strongly dependent on temperature, with 20 degrees C induction being optimal. Use of 2 micro multicopy expression constructs elevated yields over a low-copy CEN-based system by approximately 2-fold. Yeast-enhanced GFP was also expressed as a fusion to the Aga2p mating agglutinin in order to test the secretory processing fidelity of yEGFP-protein fusions. When the cell surface anchoring protein, Aga1p, was co-overexpressed with the Aga2p-yEGFP fusion, the Aga2p-yEGFP protein was tethered to the yeast cell surface. Flow cytometry and fluorescence microscopy analysis indicated that the fusion was displayed on the yeast cell surface at high levels. In the absence of high level Aga1p expression, the Aga2p-yEGFP fusion protein was instead secreted in its entirety with no detectable surface display. These findings reveal that yeast is a suitable host for secretion of GFP and GFP-protein fusions and thus could enable a wide range of biochemistry and biotechnology applications.

Development of a yeast biosensor–biocatalyst for the detection and biodegradation of the organophosphate paraoxon

Organophosphate (OP) poisoning can occur through unintentional exposure to OP pesticides, or by the deliberate release of OP nerve agents. Consequently, there is considerable interest in the development of systems that can detect and/or biodegrade these agents. The aim of this study was to generate a prototype fluorescent reporter yeast biosensor that could detect and biodegrade the model OP pesticide, paraoxon, and subsequently detect paraoxon hydrolysis. Saccharomyces cerevisiae was engineered to hydrolyze paraoxon through the heterologous expression of the Flavobacterium species opd (organophosphate degrading) gene. Global transcription profiling was subsequently used to identify yeast genes, which were induced in the presence of paraoxon, and genes, which were associated with paraoxon hydrolysis. Paraoxon-inducible genes and genes associated with paraoxon hydrolysis were identified. Candidate paraoxon-inducible promoters were cloned and fused to the yeast-enhanced green fluorescent protein (yEGFP), and candidate promoters associated with paraoxon hydrolysis were fused to the red fluorescent protein (yDsRed). The ability of the yeast biosensor to detect paraoxon and paraoxon hydrolysis was demonstrated by the specific induction of the fluorescent reporter (yEGFP and yDsRed, respectively). Biosensors responded to paraoxon in a dose- and time-dependent manner, and detection was rapid (15 to 30 min). yDsRed induction occurred only in the recombinant opd(+) strains suggesting that yDsRed induction was strictly associated with paraoxon hydrolysis. Together, these results indicate that the yeast biocatalyst-biosensor can detect and degrade paraoxon and potentially also monitor the decontamination process.

Construction and real-time RT-PCR validation of Candida albicans PALS-GFP reporter strains and their use in flow cytometry analysis of ALS gene expression in budding and filamenting cells

The gene encoding yeast-enhanced green fluorescent protein (GFP) was placed under control of ALS gene promoters in Candida albicans. The PALS-GFP reporter strains were validated using various techniques including a new real-time RT-PCR assay to quantify ALS gene expression. The PALS-GFP reporter strains were grown in media that promoted yeast or germ tube forms, and the resulting fluorescence was measured by flow cytometry. In addition to results that indicate differences in ALS gene expression due to growth medium, growth stage and developmental programme, new data show large differences in transcriptional level among the ALS genes. Expression of ALS1 was associated with transfer of the PALS1-GFP strain to fresh growth medium. ALS3 expression increased markedly when germ tubes were visible microscopically and ALS7 expression exhibited a transient peak between 2 and 3 h following inoculation into fresh YPD medium. Transcription from the ALS1 and ALS3 promoters was strongest among those tested and contrasted markedly with the weaker promoter strength at the ALS5, ALS6, ALS7 and ALS9 loci. These weaker transcriptional responses were also observed using real-time RT-PCR measurements on wild-type C. albicans cells. Assuming a positive correlation between transcriptional level and protein production, these results suggest that some Als proteins are abundant on the C. albicans cell surface while others are produced at a much lower level.

Integrative, multifunctional plasmids for hypha-specific or constitutive expression of green fluorescent protein in Candida albicans.

The authors have engineered plasmid constructs for developmental and constitutive expression of yeast-enhanced green fluorescent protein (yEGFP3) in Candida albicans. The promoter for the hyphae-specific gene Hyphal Wall Protein 1 (HWP1) conferred developmental expression of yEGFP3 in germ tubes and hyphae but not in yeasts or pseudohyphae when targeted to the ENO1 (enolase) locus in single copy. The pHWP1GFP3 construct allows for the easy visualization of HWP1 promoter activity in individual cells expressing true hyphae without having to prepare RNA for analysis. Constitutive expression of yEGFP was seen in all cell morphologies when the HWP1 promoter was replaced with the ENO1 promoter region. The use of the plasmids for expression of genes other than yEGFP3 was examined by substituting the putative C. albicans BCY1 (SRA1) gene, a component of the cAMP signalling pathway involved in yeast to hyphae transitions, for yEGFP3. Strains overexpressing BCY1 from the ENO1 promoter were inhibited in germ tube formation and filamentation in both liquid and solid media, a phenotype consistent with keeping protein kinase A in its inactive form by association with Bcy1p. The plasmids are suitable for studies of germ tube induction or assessing germ tube formation by measuring yEGFP3 expression, for inducible expression of genes concomitant with germ tube formation by the HWP1 promoter, for constitutive expression of genes by the ENO1 promoter, and for expressing yEGFP3 using a promoter of choice.

Vectors designed for efficient molecular manipulation in Candida albicans

Functional studies on genes of Candida albicans have been hampered by the fact that few vectors are available for efficient cloning and expression in C. albicans, in contrast to Saccharomyces cerevisiae. Here we report that six vectors were constructed for molecular manipulation in C. albicans. All of them contained the autonomous replicating sequence ARS2 and the uracil gene as a selective marker. Introduction of multicloning site (MCS) facilitated directional cloning into various convenient restriction sites is discussed. Distal to the MCS, the additions of sequences encoding yeast-enhanced green fluorescent protein 3 (yEGFP3) and the terminator of chitin synthase 2 (TCHS2) enabled us to express an open reading frame (ORF) with its own promoter as a GFP fusion protein, so that its intracellular localization could be easily determined. A vector of 7.4 kb was also constructed to express a cloned ORF as a GFP fusion protein under the control of an inducible MET3 promoter (PMET3) located proximal to the MCS. Since this vector was relatively large in size for expressing ORFs, two additional vectors of 6.7 kb were constructed by inserting PMET3 and TCHS2 proximal and distal to the MCS of the above vector containing MCS only, respectively. These six vectors made it possible to study C. albicans in greater detail. They can be used in identification of a promoter, intracellular localization of a protein, and in the induction of lethal genes.

The Caenorhabditis elegans Y87G2A.14 Nudix hydrolase is a peroxisomal coenzyme A diphosphatase

BackgroundThe number of Nudix hydrolase family members varies widely among different organisms. In order to understand the reasons for the particular spectrum possessed by a given organism, the substrate specificity and function of different family members must be established.ResultsThe Y87G2A.14 Nudix hydrolase gene product of Caenorhabditis elegans has been expressed as a thioredoxin fusion protein in Escherichia coli and shown to be a CoA diphosphatase with catalytic activity towards CoA and its derivatives. The products of CoA hydrolysis were 3',5'-ADP and 4'-phosphopantetheine with Km and kcat values of 220 μM and 13.8 s-1 respectively. CoA esters yielded 3',5'-ADP and the corresponding acyl-phosphopantetheine. Activity was optimal at pH 9.5 with 5 mM Mg2+ and fluoride was inhibitory with a Ki of 3 μM. The Y87G2A.14 gene product has a potential C-terminal tripeptide PTS1 peroxisomal targeting signal – SKI. By fusing a Y87G2A.14 cDNA to the C-terminus of yeast-enhanced green fluorescent protein, the enzyme appeared to be targeted to peroxisomes by the SKI signal when transfected into yeast cells. Deletion of SKI abolished specific targeting.ConclusionsThe presence of related sequences with potential PTS1 or PTS2 peroxisomal targeting signals in other organisms suggests a conserved peroxisomal function for the CoA diphosphatase members of this group of Nudix hydrolases.

The NADH Diphosphatase Encoded by the Saccharomyces cerevisiae NPY1 Nudix Hydrolase Gene Is Located in Peroxisomes

The NPY1 nudix hydrolase gene of Saccharomyces cerevisiae has been cloned and shown to encode a diphosphatase (pyrophosphatase) with NADH as the preferred substrate, giving NMNH and AMP as products. NADPH, diadenosine diphosphate, NAD+, NADP+, and ADP-ribose were also utilized efficiently. Km values for NADH, NAD+, and ADP-ribose were 0.17, 0.5, and 1.3 mM and kcat values 1.5, 0.6, and 0.6 s−1, respectively. NPY1 has a potential C-terminal tripeptide PTS1 peroxisomal targeting signal (SHL). By fusing NPY1 to the C-terminus of yeast-enhanced green fluorescent protein, the enzyme was found to be targeted to peroxisomes. Colocalization with peroxisomal thiolase was also shown by indirect immunofluorescence. Related sequences in other organisms also have potential PTS1 signals, suggesting an important peroxisomal function for this protein. This function may be the regulation of nicotinamide coenzyme concentrations independently of those in other compartments or the elimination of oxidized nucleotide derivatives from the peroxisomal environment.