Functional analysis of alcohol dehydrogenase (ADH) genes in Pichia pastoris
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Pichia
Alcohol oxidoreductase
The yeast Pichia pastoris was transformed by the multi-copy Pichia expression vector that can express secreted human albumin. The high level expression of cell line was selected after screening. The expression of human recombinant albumin in Pichia pastoris induced by different methods were compared. The ratio of secreted human albumin is 80% in total secreted proteins and the expression level reaches as high as is 10 g/L.
Pichia
Human serum albumin
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Objective:To express and identify Annexin32 in meythylotrophic yeast Pichia pastoris .Methods:Nine high copy number transformants of Pichia pastoris ,whose phenotypes were screened by culture and PCR,were induced by methanol in BMMY,BMM and MM for the expression of Annexin32.Supernatants after induction were analyzed by SDS PAGE and Western blot.The Annexin32 were separated and purified by (NH 4) 2SO 4+PI and Superdex75.The pure Annexin32 were then immunized to mice.Results:Two Mut s transformants gave high level expression in BMMY.The expression product had 2 bands (MW 38 000 and 40 000) and the concentration was 200 350 mg/L,accounting for over 80% of the total secreted protein.Our data showed higher immunogenicity in Annexin32 from yeast than from E.coli .Conclusion:Annexin32 is expressed successfully in mythylotrophic yeast Pichia pastoris ,providing a foundation for further research.
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Objective To express the kringle 5 gene of human plasminogen (hK5) in Pichia pastoris and determine the antitumor activity of expressed hK5 protein. Methods Design and synthesize hK5 gene according to the codon bias of Pichia pastoris and insert into plasmid p819. Transform the constructed recombinant plasmid p819-8α-hK5 to Pichia pastoris GS115, screen transformants by G418 pressure screening for expression under induction of methanol. The condition for fermentation culture of recombinant Pichia pastoris in shaking flask was optimized. The expressed hK5 protein was purified by DEAE-Sepharose chromatography and G75 gel filtration, and evaluated for inhibitory activity to H22 tumor in KM mouse model. Results Two transformants for high expression of hK5 protein was screened. The expression level of hK5 protein in Pichia pastoris cultured in shaking flask under the optimized condition was more than 150 mg / L. The expressed hK5 protein reached a purity of more than 95. 0% after purification and inhibited the growth of H22 tumor in KM mice. Conclusion hK5 protein was highly expressed in Pichia pastoris and showed good antitumor activity.
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The fusion gene of thanatin-linker-ppa is cloned into the Pichia pastoris expression vector.The pPlC3.5K/Thanatin-(G8S2)-PPA yeast expression vector is constructed and then sequenced.The pPIC3.5K/Thanatin-(G8S2)-PPA is linearized by Sal I enzyme,and transformed into GS115 Pichia pastoris using electricity.Then the transformants are identified by PCR and selected with high G418 resistance on a YPD plate.The positive Pichia pastorist transformants of pPlC3.5K/Thanatin-(G8S2)-PPA are induced by methynol.SDS-PAGE analysis and western bloting show that fusion protein is attained,and its molecular weight is about 31 kD.The anti-tumor activity of fusion protein,which is purified by Ni-NTA affinity chromatography,is analyzed by MTT,and the results indicate that fusion protein exhibits significant antitumor activity.
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Pichia
Lipoprotein-associated phospholipase A2
Cloning (programming)
Phospholipase A
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To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.
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The first full length IgG produced in Pichia pastoris was reported in late 1980. However, use of a wild-type Pichia expression system to produce IgGs with human-like N-linked glycans was not possible until recently. Advances in glycoengineering have enabled organisms such as Pichia to mimic human N-glycan biosynthesis and produce IgGs with human glycans on an industrial scale. Since there are only a few reports of the analytical characterization of Pichia-produced IgG, we summarize the results known in this field, and provide additional characterization data generated in our laboratories. The data suggest that Pichia-produced IgG has the same stability as that produced in Chinese hamster ovary (CHO) cells. It has similar aggregation profiles, charge variant distribution and oxidation levels as those for a CHO IgG. It contains human N-linked glycans and O-linked single mannose. Because of the comparable biophysical and biochemical characteristics, glycoengineered Pichia pastoris is an attractive expression system for therapeutic IgG productions.
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Objective To construct an Pichia pastoris expressing vector pPIC9-ZEN-jjm and screen the strains which could express high-level active proteins.Method ZEN-jjm gene was spliced into pPIC9 after digested with EcoRⅠand NotⅠ,then transformed into Pichia pastoris GS115 by electroporation.The recombinant yeast strains were screened with RDB medium and methanol inducible expression.The ZEN degradation capabilities of expressed supernatant was verified by HPLC test.Result DNA sequencing demonstrated that ZEN-jjm was inserted into pPIC9.SDS-PAGE demonstrated that one yeast strain with high-level expression was obtained,and the molecular weight of the expressed protein was about 29 kDa.The HPLC result showed that the expressed protein could effectively degrade ZEN.Conclusion ZEN-degrading enzyme is highly expressed in Pichia pastoris.
Pichia
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Ethanol metabolism
Liquid diet
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Alcohol Oxidation
Primary (astronomy)
Alcohol oxidoreductase
Primary alcohol
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