Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
 6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glyceraldehyde-3-phosphate dehydrogenase gene of Flammulina velutipes was isolated. The complete gpd sequence (from ATG to TAA) was 1,489 bp in length and contained nine introns. The locations of these nine introns were similar to those of other basidiomycetes, which might reflect the evolutionary divergence of these mushrooms. The F. velutipes gpd gene was found to encode a protein of 339 amino acids and its putative amino acid sequence revealed a high similarity to an analogous protein deriving from other basidiomycetes. Results of Southern blot analysis suggested that there existed only one copy of the gpd gene in the genome of F. velutipes and that there was one typical TATA box and two CAAT boxes located in the 5' flanking region. The F. velutipes gpd promoter was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selection marker. Using the resulting construction, hph was efficiently transformed into F. velutipes by basidiospore electroporation. No false-positive antibiotic-resistant cultures were detected by PCR amplification and the hygromycin resistance trait was maintained stably during mitotic cell division for 3 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. This rapid and convenient electroporation procedure offers new prospects for the genetic manipulation and a tool for tagging genes of this important edible mushroom species. Sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AF515622.
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PMID:Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes. 1516 94

We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.
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PMID:Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion. 1617 7

Nuclear localization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in the process of apoptosis. To study the function of GAPDH, we expressed GAPDH C-terminally fused with or without nuclear localization signal (NLS) in SH-SY5Y and NB41A3 cells using a retrovirus expression system. GAPDH carrying NLS (GAPDH-NLS) was expressed mainly in the nucleus. However, expression of GAPDH-NLS did not cause any difference in cell survival rate as compared to that of the vector alone or GAPDH without NLS. Treatment with 1-Methyl-4-phenyl-pyridium iodide (MPP+) caused no difference in the cell survival rate or in the pattern or extent of apoptosis among the three transductants. In the cells expressing GAPDH without NLS, MPP+ did not cause visible translocation of GAPDH into nucleus before the onset of apoptosis. Since GAPDH is known to comprise a CRM1-mediated nuclear export signal, we blocked the nuclear export of GAPDH by treatment with leptomycin B, an inhibitor of CRM1-mediated nuclear export. The treatment did not cause any difference in apoptosis among the three transductants. An additional treatment with MPP+ induced no apoptotic difference in these cells. Thus, we have concluded that a simple nuclear localization of GAPDH does not induce apoptosis, and that MPP+-induced apoptosis is not caused by nuclear translocation of GAPDH.
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PMID:Nuclear localization of glyceraldehyde-3-phosphate dehydrogenase is not involved in the initiation of apoptosis induced by 1-Methyl-4-phenyl-pyridium iodide (MPP+). 1632 57

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.
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PMID:Construction of a library of human glycosyltransferases immobilized in the cell wall of Saccharomyces cerevisiae. 1693 46

Tumour-specific chromosomal rearrangements are known to create chimaeric products with the ability to generate many human cancers. hTAF(II)68-TEC (where hTAF(II)68 is human TATA-binding protein-associated factor II 68 and TEC is translocated in extraskeletal chondrosarcoma) is such a fusion product, resulting from a t(9;17) chromosomal translocation found in extraskeletal myxoid chondrosarcomas, where the hTAF(II)68 NTD (N-terminal domain) is fused to TEC protein. To identify proteins that control hTAF(II)68-TEC function, we used affinity chromatography on immobilized hTAF(II)68 (NTD) and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS and isolated a novel hTAF(II)68-TEC-interacting protein, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). GAPDH is a glycolytic enzyme that is also involved in the early steps of apoptosis, nuclear tRNA export, DNA replication, DNA repair and transcription. hTAF(II)68-TEC and GAPDH were co-immunoprecipitated from cell extracts, and glutathione S-transferase pull-down assays revealed that the C-terminus of hTAF(II)68 (NTD) was required for interaction with GAPDH. In addition, three independent regions of GAPDH (amino acids 1-66, 67-160 and 160-248) were involved in binding to hTAF(II)68 (NTD). hTAF(II)68-TEC-dependent transcription was enhanced by GAPDH, but not by a GAPDH mutant defective in hTAF(II)68-TEC binding. Moreover, a fusion of GAPDH with the GAL4 DNA-binding domain increased the promoter activity of a reporter containing GAL4 DNA-binding sites, demonstrating the presence of a transactivation domain(s) in GAPDH. The results of the present study suggest that the transactivation potential of the hTAF(II)68-TEC oncogene product is positively modulated by GAPDH.
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PMID:Regulation of oncogenic transcription factor hTAF(II)68-TEC activity by human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 1730 60

The promoter of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene from Aspergillus nidulans (PgpdA) is widely used to direct expression of target genes constitutively in fungi. However, in some species, a heterogeneous promoter is found to be of low efficiency. To obtain a high-efficiency promoter for transformation of Beauveria bassiana, an entomopathogenic fungus widely used as an mycoinsecticide, a glyceraldehyde-3-phosphate dehydrogenase gene (Bbgpd) promoter, was cloned and characterized. Four deletion constructs (-2118, -1153, -726, and -354) of the 5'-upstream sequence of Bbgpd linked to a bar::gus fusion gene (phosphinothricin-resistance::beta-glucuronidase fused gene), which were used as selected marker gene and report gene, respectively, were generated. GUS activities of transgenic strains harboring -726, -1153, and -2118 deletion constructs were much stronger than that of the promoter of Aspergillus nidulans gpdA (PgpdA), with a twofold to threefold increase over that in the PgpdA construct. The -726 fragment was necessary to direct GUS expression in B. bassiana. No -354 transgenic progenies were obtained, possibly because it failed to initiate the transcription of bar::gus fusion gene. A remarkable increase of GUS activity was found between the -1153 and -726 constructs, indicating that some active transcriptional elements were located in this region. With a high expression level and relatively short sequence, PBbgpd can be used to drive target genes in B. bassiana transgenic research.
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PMID:Characterization of a highly active promoter, PBbgpd, in Beauveria bassiana. 1844 58

Short-chain esters play a significant role in the food industry as flavor and aroma constituents. Candida antarctica lipase B (CALB) is one of the most effective catalysts for organic synthesis. We constructed a CALB-displaying yeast whole-cell biocatalyst and applied it to esterification from caproic acid and ethanol. CALB was fused with the alpha-agglutinin C-terminal and the signal peptide of Glucoamylase in pICAS, a yeast surface display vector, to construct plasmid pICAS-CALB. An extremely Asn-rich linker, named celAL was inserted in the Xho I of pICAS-CALB to construct plasmid pICAS-celAL-CALB. The fused gene was under the control of GAPDH promoter. After incubated at 30 degrees C for 96 h the lipase hydrolytic activity of the yeast whole cells reached a plateau, 26.26 u/(g x dry cell). In nonaqeous media, the yield of 98.0% ethyl hexanoate was obtained after 24 h esterification from caproic acid and ethanol (the molar ratio of caproic acid : ethanol = 1 : 1.25) using lyophilized CALB displaying yeast whole cells.
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PMID:[Expression of Candida antarctica lipase B on yeast surface and synthesis of ethyl hexanoate catalyzed by CALB]. 1861 81

In our previous work, four secretory antigen-delivery systems based on different signal peptides (SPs) (SP(hlyA), SP(rtxA), SP(vah3) and SP(empA)) have been successfully established in attenuated Vibrio anguillarum. To investigate the potential application of the antigen-delivery systems in V. anguillarum multivalent vector vaccine, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from pathogenic Aeromonas hydrophila, as a putative protective antigen, was fused to the four delivery systems, respectively, and introduced into attenuated V. anguillarum strain MVAV6203 to get four GAPDH-delivery strains in this work. Immunodetection of GAPDH indicated that among the four constructs, the SP(empA)-mediated GAPDH-delivery strain AV(pGap-empA) showed the highest level of GAPDH expression and secretion, and was determined as the multivalent vaccine candidate. Further immune protection evaluation of AV(pGap-empA) in turbot (Scophtalmus maximus) demonstrated that the vaccine candidate could induce efficient protection against V. anguillarum and A. hydrophila, suggesting that this vector vaccine had great potential in serving as a valuable multivalent live vaccine.
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PMID:A novel multivalent vaccine based on secretary antigen-delivery induces protective immunity against Vibrio anguillarum and Aeromonas hydrophila. 2002 9

The aim of the study was to use Pichia pastoris to express a recombinant porcine lipase gene (pLip). The expression-secretion cassette was constructed using the P. pastoris GAPDH (glyceraldehyde-3-phosphate-dehydrogenase) promoter and an 89-residue prepro-alpha-factor secretion signal fused to the AOX1 terminator (the pGAPZalphaA vector). A total of 1,408 bp of pancreatic lipase cDNA was produced, which was located from the position of 4-nt upstream of ATG to 1408-nt inside the intact coding region of the pLip sequence. In an animal trial, three concentrations of recombinant lipase activity (0, 5,000 and 10,000 U/kg) were blended with the basal diet and fed to weaned piglets for six weeks. During the experimental period, the growth performance (bodyweight, feed intake, and feed efficiency) of the test groups was superior to that of the control group (p < 0.05). Furthermore, the group fed the diet blended with 10,000 U/kg of recombinant lipase showed significant (p < 0.05) increases in blood triglyceride (TG) concentration on the seventh day postweaning. These results suggested that the porcine lipase protein yielded by transformed yeast cells may improve fat digestibility and enhance the growth performance in postweaning piglets.
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PMID:Application of porcine lipase secreted by pichia pastoris to improve fat digestion and growth performance of postweaning piglets. 2016 58

In oxygenic photosynthetic organisms, the activities of two Calvin cycle enzymes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH and phosphoribulokinase, PRK) are regulated by CP12-mediated complex formation. The Arabidopsis genome contains three genes encoding different CP12 isoforms (CP12-1, At2g47400; CP12-2, At3g62410 and CP12-3, At1g76560), all plastid-targeted, as demonstrated by localization in the chloroplast stroma of CP12 precursor sequences fused with the green fluorescence protein (GFP). The disorder predictor PONDR classified Arabidopsis CP12s as largely disordered proteins, and circular dichroism spectra confirmed these predictions. Based on sequence similarity, 66 CP12s from different organisms were identified and clustered in six types, with CP12-1 and -2 grouping together with other largely disordered sequences (Type I), while a lower level of disorder was predicted within the cluster including CP12-3 (Type II). The three Arabidopsis CP12 isoforms were expressed as mature recombinant forms and purified to homogeneity. Redox titrations demonstrated that the four conserved cysteines of each CP12 isoform could form two internal disulfide bridges with different midpoint redox potentials (E(m,7.9) -326 mV and -350 mV in both CP12-1 and CP12-2; E(m,7.9) -332 mV and -373 mV in CP12-3). In agreement with their similar redox properties, all CP12 isoforms formed, in vitro, a supramolecular complex with GAPDH and PRK, with comparable inhibitory effects on both enzyme activities. In order to test whether CP12 isoforms might have broader regulatory functions than regulating Calvin cycle enzymes, CP12 proteins were analyzed for their capacity to bind plastidial glycolytic GAPDH (GapCp). To this purpose, the mature form of Arabidopsis GapCp2 was cloned, expressed in recombinant form and purified to homogeneity. However, contrary to expectations, no CP12 isoform was able to bind GapCp2 under any of the conditions tested.
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PMID:In vitro characterization of Arabidopsis CP12 isoforms reveals common biochemical and molecular properties. 2039 32


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