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Yeast Pichia Pastoris (yeast + pichia_pastori)

Distribution by Scientific Domains

Life Sciences	55%
Chemistry	33%

Kinds of Yeast Pichia Pastoris

methylotrophic yeast pichia pastori

Selected Abstracts

Expression of Secreted His-Tagged S -adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and Immobilization

BIOTECHNOLOGY PROGRESS, Issue 1 2008
Yunxing Luo
S -Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S -adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomycescerevisiae was successfully produced at high level (,200 mg/L) by the recombinant methylotrophic yeast Pichiapastoris. The secreted His6 -tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 °C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl -Met and ATP. [source]

Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in the Yeast Pichia pastoris

BIOTECHNOLOGY PROGRESS, Issue 5 2003
Lina Gu
A gene encoding manganese peroxidase (mnp1) from Phanerochaetechrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P.chrysosporium fungal secretion signal, p,AMNP contains an ,-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and ,-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55,100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous MnII, CaII, and FeIII conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 °C. [source]

Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris

FEBS JOURNAL, Issue 6 2000
Ludovic Otterbein
Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae,-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated. [source]

Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells

PARASITE IMMUNOLOGY, Issue 3 2007
H. S. GOODRIDGE
SUMMARY Modulation of macrophage/dendritic cell (DC) cytokine production by the filarial nematode phosphorylcholine (PC)-containing product, ES-62, is mediated by Toll-like receptor (TLR) 4 and signal transduction depends on the TLR adaptor MyD88. Intriguingly, comparison of TLR4 knock-out (ko) mice with TLR4 mutant C3H/HeJ mice indicates that ES-62 cytokine responses are not dependent on the Pro712 residue of TLR4, which is crucial for the response to bacterial lipopolysaccharide (LPS). Because other immunomodulatory effects of ES-62 have been attributed to PC we have now investigated, using PC conjugated to ovalbumin (PC-Ova), whether PC is responsible for the interaction of ES-62 with TLR4. PC-Ova mimicked the modulation of interleukin (IL)-12 production by ES-62 in a TLR4- and MyD88-dependent manner and as with native ES-62, PC-Ova effects were not dependent on Pro712. Furthermore, both native ES-62 and PC-Ova suppressed Akt phosphorylation, whereas neither altered the activation of p38 or Erk MAP kinases. To rule out any role for the ES-62 protein component, we tested a PC-free recombinant ES-62 (rES-62) generated in the yeast Pichia pastoris. Surprisingly, rES-62 also modulated IL-12 production, but in a TLR4/MyD88-independent manner. Furthermore, rES-62 strongly activated both the p38 and Erk MAP kinases and Akt. However, recent biophysical analysis suggests there are differences in folding/shape between native and rES-62 and hence data obtained with the latter should be treated with caution. Nevertheless, although our study indicates that PC is likely to be primarily responsible for the modulation of cytokine production observed with native ES-62, an immunomodulatory role for the protein component cannot be ruled out. [source]

NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditions

PLANT BIOTECHNOLOGY JOURNAL, Issue 6 2010
Roxana Portieles
Summary Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions. [source]

Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastoris

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2000
Glenn E. Dale
Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor. The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris. The protein was purified to homogeneity and single crystals have been obtained. Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP,phosphoramidon binary complex and the apo enzyme. [source]

Crystallization of Saccharomyces cerevisiae,-mannosidase, a cargo protein of the Cvt pathway

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009
Yasunori Watanabe
Saccharomyces cerevisiae,-mannosidase (Ams1) is a cargo protein that is transported to the vacuole by the cytoplasm-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ams1 functions as a resident hydrolase. Ams1 has been overexpressed in the methylotrophic yeast Pichia pastoris, purified and crystallized in two crystal forms. Form I belongs to space group P21, with unit-cell parameters a = 145.7, b = 127.7, c = 164.0,Å, , = 101.5°. Form II belongs to space group I222 or I212121, with unit-cell parameters a = 127.9, b = 163.7, c = 291.5,Å. Diffraction data were collected from these crystals to a resolution of 3.3,Å for form I and of 2.6,Å for form II using synchrotron radiation. [source]

Optimization of the expression of recombinant human activin A in the yeast Pichia pastoris

BIOTECHNOLOGY PROGRESS, Issue 2 2010
Dale Fredericks
Abstract We report a new procedure to express recombinant human activin A using the methanolic yeast, Pichia pastoris. Optimization of culture procedures has involved comprehensive examination of the effects of culture vessel shape, volume of broth in the induction and expression cultures, methanol concentration, culturing temperature, and pH of the expression cultures. After this optimization, as well as modification of the native cleavage sites, a laboratory scale procedure has been established which routinely produced 2,10 mg/L amounts of this vital growth factor in the highly efficient, eukaryotic yeast system. This system avoids the need to produce this protein and similar TGF-, proteins in mammalian cell lines which, in addition to being costly, produce many native binding partners of these cystine knot proteins, a factor which can dramatically affect yields of the target protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]