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D R Higgins - One of the best experts on this subject based on the ideXlab platform.
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Overview of protein expression in Pichia pastoris.
Current protocols in protein science, 2001Co-Authors: D R HigginsAbstract:Pichia pastoris is a methylotrophic yeast and can be used as a heterologous expression system. This microorganism is as easy to manipulate as Escherichia coli, but has many of the advantages of eukaryotic expression (e.g., protein processing, folding, and post-translational modifications), and it is faster, easier, and cheaper to use than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture. It also generally yields higher expression levels. This overview discusses important considerations for the use of Pichia pastoris, including strains for expression, expression plasmids, transformation by integration, and post-translational modifications. Examples of expression are given and finally, legal issues regarding patent rights for heterologous protein expression in Pichia pastoris are described.
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Current Protocols in Protein Science - Overview of protein expression in Pichia pastoris.
Current Protocols in Protein Science, 1995Co-Authors: D R HigginsAbstract:Pichia pastoris is a methylotrophic yeast and can be used as a heterologous expression system. This microorganism is as easy to manipulate as Escherichia coli, but has many of the advantages of eukaryotic expression (e.g., protein processing, folding, and post-translational modifications), and it is faster, easier, and cheaper to use than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture. It also generally yields higher expression levels. This overview discusses important considerations for the use of Pichia pastoris, including strains for expression, expression plasmids, transformation by integration, and post-translational modifications. Examples of expression are given and finally, legal issues regarding patent rights for heterologous protein expression in Pichia pastoris are described.
Thomas S. Vedvick - One of the best experts on this subject based on the ideXlab platform.
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Gene expression in yeast: Pichia pastoris.
Current opinion in biotechnology, 1991Co-Authors: Thomas S. VedvickAbstract:Recent studies have shown the versatility and utility of the Pichia pastoris expression system. Improvements in strains have boosted the yield of proteins and peptides to the commercially feasible range. The Pichia pastoris expression system will soon be used to manufacture proteins for human clinical trials.
Richard D. Braatz - One of the best experts on this subject based on the ideXlab platform.
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Modeling of copy number variability in Pichia pastoris.
Biotechnology and bioengineering, 2021Co-Authors: Andrew J. Maloney, Neil C. Dalvie, Joseph R. Brady, Kerry R. Love, J. Christopher Love, Richard D. BraatzAbstract:Development of continuous biopharmaceutical manufacturing processes is an area of active research. This study considers the long-term transgene copy number stability of Pichia pastoris in continuous bioreactors. We propose a model of copy number loss that quantifies population heterogeneity. An analytical solution is derived and compared with existing experimental data. The model is then used to provide guidance for stable operating timescales. The model is extended to consider copy number dependent growth such as in the case of Zeocin supplementation. The model is also extended to analyze a continuous seeding strategy. This study is a critical step towards understanding the impact of continuous processing on the stability of Pichia pastoris and the resultant products.
Andrew J. Maloney - One of the best experts on this subject based on the ideXlab platform.
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Modeling of copy number variability in Pichia pastoris.
Biotechnology and bioengineering, 2021Co-Authors: Andrew J. Maloney, Neil C. Dalvie, Joseph R. Brady, Kerry R. Love, J. Christopher Love, Richard D. BraatzAbstract:Development of continuous biopharmaceutical manufacturing processes is an area of active research. This study considers the long-term transgene copy number stability of Pichia pastoris in continuous bioreactors. We propose a model of copy number loss that quantifies population heterogeneity. An analytical solution is derived and compared with existing experimental data. The model is then used to provide guidance for stable operating timescales. The model is extended to consider copy number dependent growth such as in the case of Zeocin supplementation. The model is also extended to analyze a continuous seeding strategy. This study is a critical step towards understanding the impact of continuous processing on the stability of Pichia pastoris and the resultant products.
Huang Jian - One of the best experts on this subject based on the ideXlab platform.
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Expression of soybean peroxidase from soybean in Pichia pastoris
Microbiology China, 2014Co-Authors: Huang JianAbstract:[Objective] Soybean peroxidase(SBP) will be widely used in immunoassay, and wastewater treatment and so on, due to its wide substrates, high specific activity, and good thermal stability. Nowadays, it was obtained mainly by extracted from soybean hull. However, it cannot meet the requirements of industrial applications for its low yield, high cost. In this study, SBP will be expressed in Pichia pastoris. [Methods] Both the genes of SBP and truncated C-terminal 20 amino acid SBP were cloned into pPIC-9K. These constructed expression vectors were transformed into Pichia pastoris X-33, and then be used to express SBP. Furthermore, the effects of asparagine glycosylation on SBP expression were also investigated by mutating asparagine into glutamine.[Results] Full length of SBP is inactive in Pichia pastoris. But the truncated C-terminal 20 amino acid SBP showed 23.5 U/mL. Our results indicated that glycosylation site of 144, 185, 197 have a great effect on the enzyme activity. These mutants were almost inactive; Whereas 211 and 216 deglycosylation sites had little effect on activity of SBP, can not be deglycosylation. [Conclusion]The highest activity of SBP was 510 U/mL in fermentation, which is the highest level of the reported.
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Intracellular expression of rPA gene in Pichia pastoris
Chinese Journal of Biochemical Pharmaceutics, 2003Co-Authors: Huang JianAbstract:Purpose To construct a recombinant plasmid pPIC9k rPA for the expression of whole length of rPA gene in Pichia pastoris. MethodsThe plasmid pJZ16 containing the whole length of rPA gene was digested with Bam H Ⅰ and Not Ⅰ and cloned into expression vector pPIC9k. Identified by sequence detemination and then transformed by electroporating.The positive transformants with the integrated rPA gene were identified by PCR and expressed in Pichia pastoris yeast cells. The expressed products were analyzed by SDS PAGE, Western blots and fibrin dissolving activity tests.ResultsThe expression of intracellular, insoluble, unglycosylated rPA was obtained. SDS PAGE showed a relative molecular weight of expressed protein of about 39 000 and Western blots showed the specific reaction of it with McAb. When dissolved in 8M urea, the inclusion body rPA exhibited fibrin dissolving activity.ConclusionThe recombinant rPA protein was successfully expressed in Pichia pastoris .