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Methylotrophic Yeast Pichia Pastoris (methylotrophic + yeast_pichia_pastori)
Selected AbstractsExpression of Secreted His-Tagged S -adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and ImmobilizationBIOTECHNOLOGY PROGRESS, Issue 1 2008Yunxing 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] Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastorisACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2000Glenn 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 pathwayACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Yasunori 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] Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in the Yeast Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 5 2003Lina 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] | ||||||||||