A novel multi-enzymatic high throughput assay for transaminase activity

Weinhandl, Katrin; Winkler, Margit; Glieder, Anton; Camattari, Andrea

doi:10.1016/j.tet.2012.06.058

Cited by 18 publications

(9 citation statements)

References 22 publications

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“…The majority of transformants will have a single copy of the expression vector integrated into the genome, and numerous clones will have to be screened to find high-copy transformants (Lin-Cereghino and Lin-Cereghino 2007 ). Therefore, several high-throughput methods have been established to screen a large number of clones based on small-scale cultivation in deep well plates (Mellitzer et al 2012 ; Weinhandl et al 2012 ; Weis et al 2004 ). The selected clones, however, might not perform as well in fermenter cultivations due to different cultivation conditions.…”

Section: Expression Strategies and Industrial Applicationsmentioning

confidence: 99%

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

819

564

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Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

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Section: Expression Strategies and Industrial Applicationsmentioning

confidence: 99%

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

819

564

View full text Add to dashboard Cite

show abstract

“…In recent years, screening of microorganisms in general has benefited from a number of technological advances, allowing expansion, even in very simple laboratory settings, of the number of parallel batch-type experiments that can be carried out using 96-deep-well plates or similar devices (Camattari et al, 2014, Weinhandl et al, 2012, Weis et al, 2004.…”

Section: Screening In Batch Modementioning

confidence: 99%

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Looser

Brühlmann

Bumbak

et al. 2015

Biotechnology Advances

295

268

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Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

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“…Pada umumnya transforman hanya memiliki satu salinan gen yang terintegrasi ke dalam genom inang sehingga banyak klon yang harus diseleksi untuk mendapatkan klon transforman multikopi (Lin-Cereghino dan Lin- Cereghino, 2007). Untuk mempercepat mendapatkan transforman multikopi, beberapa metode terbaru (high-throughput) telah digunakan untuk menskrining klon dalam jumlah yang banyak melalui kultur skala kecil pada deep well plates (Mellitzer et al, 2012;Weinhandl et al, 2012;Weis et al, 2004).…”

Section: Pentingnya Pengembangan Transforman Yang Diinginkanunclassified

Pichia pastoris: SEL RAGI UNTUK PRODUKSI PROTEIN REKOMBINAN

2018

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Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein production which is widely used for biopharmaceutical-based drug production. This yeast can grow fast with a high cell density. Its genetic stability, high cell density, and stress resistance make the development process and scale-up of P. pastoris can increase to a scale of 200,000 liters of culture. In contrast to the expensive and complex development of recombinant protein production in mammalian cells, the development of production in P. pastoris is relatively simple and cheaper. The advantage of P. pastoris as an expression system is that it is able to use methanol as a carbon source by inducing the expression of alcohol oxidase oxidase (AOX) enzyme. Promoter used by this enzyme is also used as a strong promoter for the expression of proteins that we want. Unlike in bacterial and mammalian systems, recombinant protein production in Pichia cells is not contaminated with endotoxins or viruses so it is safer and simplifies the downstream processes in bioproduction. The level of endogenous protein in the low supernatant allows Pichia to cultivate with a high volumetric productivity, therefore the process of protein production becomes very economical. This review provides an overview of several things that must be considered in utilizing P. pastoris as an expression system including the selection of vectors, strains, vector integration mechanisms into the genome, glycosylation processes, and applications in industry.

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A novel multi-enzymatic high throughput assay for transaminase activity

Cited by 18 publications

References 22 publications

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Pichia pastoris: SEL RAGI UNTUK PRODUKSI PROTEIN REKOMBINAN

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