Topic
Pichia pastoris
About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.
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TL;DR: Using RT-PCR and rapid amplication of cDNA ends (RACE) techniques, a 1345bp full-length cDNA fragment was obtained from Aspergillus sulphureus and the recombinant beta-mannanase gene was successfully expressed in a fully active form in Pichia pastoris.
129 citations
TL;DR: Amperometric alcohol biosensors were constructed by co-immobilising commercially available alcohol oxidase with the hydrogen peroxide reducing enzyme, horseradish peroxidase, in a carbon paste matrix and found to yield the best characteristics (sensitivity, selectivity, operational and storage stability, ethanol conversion).
128 citations
Book•
01 Jan 2004
TL;DR: Protein expression in mammalian cells expression in Xenopus oocytes and cell-free extracts and cloned genes in the yeasts Saccharomyces cerevisiae and Pichia pastoris Baculovirus expression systems.
Abstract: Protein expression in mammalian cells expression in Xenopus oocytes and cell-free extracts Expressing cloned genes in the yeasts Saccharomyces cerevisiae and Pichia pastoris Baculovirus expression systems protein synthesis in eukaryotic cell-free systems Prokaryotic in vivo expression systems cell-free coupled transcription-translation systems from Escherischia Coli monitoring protein expression
128 citations
TL;DR: A set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.
Abstract: Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. glucose) limitation. DNA microarray analysis of P. pastoris wild type cells growing in glycerol-based batch and glucose-based fed batch was used for the identification of genes with both, strong repression on glycerol and high-level expression in the feed phase. Six novel glucose-limit inducible promoters were successfully applied to express the intracellular reporter eGFP. The highest expression levels together with strong repression in pre-culture were achieved with the novel promoters PG1 and PG6. Human serum albumin (HSA) was used to characterize the promoters with an industrially relevant secreted protein. A PG1 clone with two gene copies reached about 230% of the biomass specific HSA titer in glucose-based fed batch fermentation compared to a PGAP clone with identical gene copy number, while PG6 only achieved 39%. Two clones each carrying eleven gene copies, expressing HSA under control of PG1 and PG6 respectively were generated by post-transformational vector amplification. They produced about 1.0 and 0.7 g L-1 HSA respectively in equal fed batch processes. The suitability in production processes was also verified with HyHEL antibody Fab fragment for PG1 and with porcine carboxypeptidase B for PG6. Moreover, the molecular function of the gene under the control of PG1 was determined to encode a high-affinity glucose transporter and named GTH1. A set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.
127 citations
01 Jan 1999
TL;DR: This book discusses how to select a Suitable Expression System for Protein Expression in Genomics and the Future of Protein Expression Systems, and some examples of systems used in the past and those currently in use.
Abstract: Contributors. Introduction: The Art of Expression. Purpose of This Book. Selecting a Suitable Expression System: Considerations. Genomics and the Future of Protein Expression Systems, J.M. Fernandez and J.P. Hoeffler. Prokaryotic Expression Systems: Gene Expression Systems Based on Bacteriophage T7 RNA Polymearse, R. Durbin. Expression Vectors Employing the trc Promoter, J. Brosius. Bacillus Expression: A Gram Positive Model, E. Ferrari and B. Miller. araB Expression System in Escherichia coli, M. Better. Eukaryotic Expression Systems: Adenoviral Vectors for Protein Expression, D.J. von Seggern and G.R. Nemerow. Expression in the Methalotrophic Yeast Pichia pastoris, J.M. Cregg. Recombinant Protein Expression in Pichia methanolica, C.K. Raymond. Cytomegalovirus Promoter for Expression in Mammalian Cells, M.F. Stinski. Inducible Mammalian Expression Systems, M.Russell. Protein Expression in Mammalian Cells Using Sindbis Virus, R.P. Bennett. Expression in Insect Systems: Drosophila S2 System for Heterologous Gene Expression, R.B. Kirkpatrick and A. Shatzman. Baculovirus Expression Vector System, M. Galleno and A.J. Sick. Transgenic Expression: Recombinant Protein Expression in Transgenic Mice, R. Abbud and J.H. Nilson. Expression of Recombinant Proteins in the Milk of Transgenic Animals, H.M. Meade, Y. Echelard, C.A. Ziomek, M.W. Young, M. Harvey, E.S. Cole, S. Groet, T.E. Smith, and J.M. Curling. Recombinant Protein Expression in Plants, A.E. Voloudakis, Y. Yin, and R.N. Beachy. Index.
127 citations