Methanol Induction Optimization for Recombinant Human Consensus Interferon Mutant Production in Pichia pastoris

2010 ◽  
Vol 150 ◽  
pp. 540-540
Author(s):  
Dan Wu ◽  
Ju Chu ◽  
Yu-You Hao ◽  
Yong-Hong Wang ◽  
Ying-Ping Zhuang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methanol Induction ◽  
Consensus Interferon

scholarly journals Improved Production ofAspergillus usamiiendo-β-1,4-Xylanase inPichia pastorisvia Combined Strategies

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Jianrong Wang ◽  
Yangyuan Li ◽  
Danni Liu
Keyword(s):  
Pichia Pastoris ◽  
Cell Viability ◽  
Recombinant Proteins ◽  
Recombinant Strain ◽  
Xylanase Activity ◽  
Vitreoscilla Hemoglobin ◽  
Wild Type ◽  
Methanol Induction ◽  
Aspergillus Usamii ◽  
Induction Temperature

A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase fromAspergillus usamii(A. usamii) inPichia pastoris(P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene fromA. usamiiwas optimized forP. pastorisand expressed inP. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression ofxynB-opt, theVitreoscilla hemoglobin(VHb) gene was transformed to the recombinant strain containingxynB-opt. The results showed that recombinant strain harboring thexynB-optandVHb(named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHbin 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHbwas optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHbreached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins inP. pastoris.

Expression and Aggregation of Recombinant Human Consensus Interferon-α Mutant by Pichia pastoris

2006 ◽  
Vol 28 (12) ◽  
pp. 905-909 ◽  
Author(s):  
Yuyou Hao ◽  
Ju Chu ◽  
Yonghong Wang ◽  
Siliang Zhang ◽  
Yingping Zhuang
Keyword(s):  
Pichia Pastoris ◽  
Interferon Α ◽  
Consensus Interferon

scholarly journals Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris

2003 ◽  
Vol 376 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Isabel SOARES-SILVA ◽  
Dorit SCHULLER ◽  
Raquel P. ANDRADE ◽  
Fátima BALTAZAR ◽  
Fernanda CÁSSIO ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Lactic Acid ◽  
Pichia Pastoris ◽  
Plasma Membranes ◽  
Membrane Vesicle ◽  
Dry Weight ◽  
Functional Expression ◽  
Acid Uptake ◽  
Methanol Induction

In Saccharomyces cerevisiae the activity for the lactate–proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a Vmax of 2.1 nmol·s−1·mg of dry weight−1. Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest Vmax (0.84 nmol·s−1·mg of dry weight−1) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.

scholarly journals Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction

2020 ◽  
Vol 27 (4) ◽  
pp. 283
Author(s):  
Patricia Gita Naully ◽  
Neni Nurainy ◽  
Elvi Restiawaty ◽  
Dessy Natalia ◽  
Debbie Soefie Retnoningrum ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Hepatitis B ◽  
Vaccine Development ◽  
Expression Cassette ◽  
Foreign Gene ◽  
Pastoris Gs115 ◽  
Methanol Induction ◽  
Small Surface ◽  
Major Health Problem ◽  
Replacement Method

Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction.

scholarly journals Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain

2020 ◽  
Vol 85 (1) ◽  
pp. 25-35
Author(s):  
Ana Balaz ◽  
Marija Blazic ◽  
Nikolina Popovic ◽  
Olivera Prodanovic ◽  
Raluca Ostafe ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phanerochaete Chrysosporium ◽  
Sodium Dodecyl ◽  
Cellobiose Dehydrogenase ◽  
Yeast Cells ◽  
Soluble Form ◽  
Kinetic Characterization ◽  
Methanol Induction ◽  
Ph Optimum ◽  
Mutant Proteins

Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZ? plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture.

Expression, Purification and Characterization of Recombinant Human Coagulation Factor XIIIa in Pichia Pastoris

2020 ◽  
Vol 27 ◽  
Author(s):  
Linyan Cheng ◽  
Ting Zhang ◽  
Yuchang Fei ◽  
Hao Shen ◽  
Hui Huang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Biological Activity ◽  
Pichia Pastoris ◽  
Critical Role ◽  
Coagulation Factor ◽  
Factor Xiiia ◽  
Expression And Purification ◽  
Methanol Induction ◽  
Healing Tissue

Background: Coagulation factor XIIIa(FXIIIa) plays a critical role in the final stage of blood coagulation. It is extremely important in wound healing, tissue repairing and promoting cell adhesion. The deficiency of the coagulation factor can cause hemorrhage and slow wound healing. Objective: In this study, recombinant pPICZαC-FXIIIa was expressed in Pichia pastoris, purified as well as its biological activity was determined. Methods: The FXIIIa fragment obtained from the human placenta was inserted into pPICZαC to obtain pPICZαC-FXIIIa, which was transformed into X33 after linearization, and FXIIIa inserted into Pichia pastoris X33 was screened for methanol induction. The expressed product was identified by western blotting, then the supernatant was purified by affinity chromatography, and the purified product was determined by plasma coagulation experiment. Results: Polymerase Chain Reaction(PCR) showed that the FXIIIa fragment of 2250 bp was inserted successfully into pPICZαC. The expression and purification products of the same molecular weight as target protein(about 83 kDa) were obtained, which solidified significantly when reacted with plasma. Conclusion: The expression and purification products were successful, with sufficient biological activity, which can be used as a candidate FXIIIa hemostatic agent in genetic engineering.

Expression of bovine interferon-γ in Escherichia coli and Pichia pastoris and comparison of their antiviral activities

2006 ◽  
Vol 3 (1) ◽  
pp. 39-46
Author(s):  
Shi Xi-Ju ◽  
Zhang Can ◽  
Han Chun-Lai ◽  
Xia Chun ◽  
Wang Ming
Keyword(s):  
Escherichia Coli ◽  
Pichia Pastoris ◽  
Cell Line ◽  
Chicken Embryo Fibroblast ◽  
Interferon Γ ◽  
Peripheral Blood Lymphocytes ◽  
Methanol Induction ◽  
E Coli ◽  
Antiviral Activities ◽  
Vesicular Stomatitis

AbstractThe bovine interferon-γ gene cloned from total RNA of peripheral blood lymphocytes stimulated with concanavalin A (ConA), using the reverse transcriptase-polymerase chain reaction (RT-PCR), was cloned into pGEM T-easy vector and sequenced. The mature peptide without signal peptide was then subcloned and constructed into the expression plasmids pET28a/BoIFN-γ and pPICZα/BoIFN-γ, respectively. The former was highly expressed, induced by isopropyl β-d-thiogalactoside (IPTG), in Escherichia coli BL21, with 30% of bacterial proteins in inclusion bodies. The latter was expressed in Pichia pastoris GS115 with methanol induction. The expressed products were secreted directly into cultural supernatant at concentrations of 1.0 g/l. The same recombinant proteins had antiviral activities 10 times higher in an MDBK (Madan-Darby bovine kidney)/VSV (Vesicular stomatitis virus) cell line than in a CEF (chicken embryo fibroblast)/VSV) cell line, and the antiviral activities expressed in P. pastoris were higher than those expressed in E. coli in the same cell line.

scholarly journals The mechanisms of fructose 1,6 bisphosphatase degradation in methylotrophic yeasts Pichia pastoris

2018 ◽  
Vol 22 ◽  
pp. 235-239
Author(s):  
O. V. Dmytruk ◽  
N. V. Bulbotka ◽  
A. A. Sibirny
Keyword(s):  
Pichia Pastoris ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Specific Activity ◽  
Wild Type ◽  
Methylotrophic Yeasts ◽  
Methanol Induction ◽  
Fructose 1,6 Bisphosphatase ◽  
In The Wild

Aim. The study of the mechanisms of fructose-1,6-bisphosphatase degradation in methylotrophic yeasts Pichia pastoris. Methods. Methods of determination the specific activity of fructose-1,6-bisphosphatase in the wild type and mutant strains of methylotrophic yeast P. pastoris after shifting cells from the medium with methanol into the medium with glucose were used. The study of fructose-1,6-bisphosphatase protein degradetion was performed by Western blot analysis. Results. The changes of the specific activity of fructose-1,6-bisphosphatase in the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of P. pastoris in short-term and long-term induction with methanol, and with or without the addition of the MG132 (proteasome degradation inhibitor) was investigated. Degradation of fructose‑1,6‑bisphosphatase by the Western blot analysis in GS200, SMD1163 and Δgss1 strains was studied. Conclusions. It was shown that the duration of cell incubation on methanol has no particular effect on the inactivation of the enzyme. The effect of the proteasome inhibitor MG132 was insignificant. Catabolic inactivation of cytosolic and peroxisomal enzymes is damaged in the Δgss1 mutant as glucose signaling is impaired. Fructose-1,6-bisphosphatase degrades by a vacuolar pathway, regardless of the duration of methanol induction, which correlates with the activity data of this enzyme. Keywords: fructose-1,6-bisphosphatase, yeasts, Pichia pastoris, methanol, autophagy.

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