scholarly journals Modified protocol for quick and large-scale transformation ofPichia pastoris

2018 ◽  
Author(s):  
Ravinder Kumar
Keyword(s):  
Pichia Pastoris ◽  
Cell Density ◽  
Large Scale ◽  
Methylotrophic Yeast ◽  
Protein S ◽  
Scale Transformation ◽  
Biological Research ◽  
Expression And Purification ◽  
Competent Cells ◽  
Resource Space

AbstractOver the last couple of decades, methylotrophic yeast,Pichia pastorisemerges as an important yeast species owing to its increasing application in industry and basic biological research. Transformation ofPichia pastoriscells for the introduction of the gene of interest is common practice for expression and purification of a heterologous protein(s). Presently available protocol ofPichia pastoristransformation involves preparation of competent cells and followed by their transformation. Preparation of competent cells requires growth of cells to certain cell density which requires lots of resource, space, time and efforts. This limits the number of transformations that can be performed by an individual at a time. In the present paper, I will describe a modification in the available protocol which makesP. pastoristransformation hassle free. The present procedure does not require growth of pre-culture or growth of cells to certain cell density rather cells are grown in a patch on YPD plate(s) and rest procedure is performed in small eppendrof tubes which allow a large number of transformations in quickest possible time with minimal resource and efforts. In the end, I also compare various protocols in tabular form which allows the user to choose best suitable procedure depending on the available resource, time, number of transformations, requirement, and efforts. The present modified protocol does not require big centrifuge and shaker which further makes this procedure more useful. I believe that present protocol of transformation with its many unique features will be really helpful to those working withP. pastoris.

scholarly journals Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

2015 ◽  
Vol 11 ◽  
pp. 1741-1748 ◽  
Author(s):  
Martina Geier ◽  
Christoph Brandner ◽  
Gernot A Strohmeier ◽  
Mélanie Hall ◽  
Franz S Hartner ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methanol Oxidation ◽  
Large Scale ◽  
Methylotrophic Yeast ◽  
Cofactor Regeneration ◽  
Regeneration System ◽  
Nadh Regeneration ◽  
Whole Cell ◽  
Knock Out ◽  
In The Wild

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.

scholarly journals Heterologous Expression of Histidine Acid Phytase From Pantoea sp. 3.5.1 in Methylotrophic Yeast Pichia Pastoris

2020 ◽  
Vol 14 (1) ◽  
pp. 179-189
Author(s):  
Aliya Suleimanova ◽  
Daria Bulmakova ◽  
Margarita Sharipova
Keyword(s):  
Pichia Pastoris ◽  
Large Scale ◽  
Residual Activity ◽  
Methylotrophic Yeast ◽  
Feed Additives ◽  
Cloning And Expression ◽  
Small Scale ◽  
Excellent Thermal Stability ◽  
Inulinase Gene ◽  
Recombinant Phytase

Background and Objective: The major storage form of phosphorus in plant-derived feed is presented by phytates and not digested by animals. Phytases are able to hydrolyze phytates and successfully used as feed additives. Nevertheless, nowadays, there is a constant search of new phytases and expression systems for better production of these enzymes. In this study, we describe cloning and expression of gene encoding histidine acid phytase from Pantoea sp. 3.5.1 using methylotrophic yeast Pichia pastoris as the host. Methods: The phytase gene was placed under the control of the methanol-inducible AOX1 promoter and expressed in P. pastoris. Experiments of small-scale phytase expression and activity assays were used to test recombinant colonies. Four different signal peptides were screened for better secretion of phytase by P. pastoris. After 36 h of methanol induction in shake flasks, the maximum extracellular phytase activity (3.2 U/ml) was observed in P. pastoris strain with integrated construct based on pPINK-HC vector and Kluyveromyces maxianus inulinase gene signal sequence. This phytase was isolated and purified using affinity chromatography. Results: Recombinant phytase was a glycosylated protein, had a molecular weight of around 90 kDa and showed maximum activity at pH 4.0 and at 50°C. Recombinant phytase had excellent thermal stability – it retained high residual activity (100% ± 2%) after 1 hour of heat treatment at 70°C. Conclusion: The enhanced thermostability of the recombinant phytase, its expression provided by strong inducible promotor and the effectively designed expression cassette, the simple purification procedure of the secreted enzyme, and the possibility of large-scale expression make the foundation for further production of this bacterial phytase in P. pastoris at an industrial scale.

High-Level Secretory Expression and Purification of Recombinant Human Interleukin 1 Beta in Pichia pastoris

2016 ◽  
Vol 23 (8) ◽  
pp. 763-769 ◽  
Author(s):  
Pengfei Li ◽  
Ganggang Yang ◽  
Xiaofang Geng ◽  
Jinbao Shi ◽  
Bin Li ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Interleukin 1 ◽  
Interleukin 1 Beta ◽  
Expression And Purification ◽  
Secretory Expression ◽  
High Level

Next-Generation Sequencing: An Emerging Tool for Drug Designing

2019 ◽  
Vol 25 (31) ◽  
pp. 3350-3357 ◽  
Author(s):  
Pooja Tripathi ◽  
Jyotsna Singh ◽  
Jonathan A. Lal ◽  
Vijay Tripathi
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Large Scale ◽  
Massively Parallel Sequencing ◽  
Genomic Research ◽  
Biological Research ◽  
Next Generation ◽  
Human Welfare ◽  
Drug Designing ◽  
Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

2014 ◽  
Vol 1841 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Lisa Klug ◽  
Pablo Tarazona ◽  
Clemens Gruber ◽  
Karlheinz Grillitsch ◽  
Brigitte Gasser ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris
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