Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

Holmes, William J.; Darby, Richard A. J.; Wilks, Martin D.B.; Smith, Rodney; Bill, Roslyn M.

doi:10.1186/1475-2859-8-35

Cited by 67 publications

(52 citation statements)

References 29 publications

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“…However, identifying a clone able to express large amounts of the desired product does not merely entail selecting the "best producer" that is appropriate for the final production/manufacturing scale (Mellitzer et al, 2012). High production is generally detrimental to cell metabolism and cell survival (Holmes et al, 2009, Jafari et al, 2011 and, thus, the "best" producers from initial screening may fail in bioreactor processes. Therefore, clustering clones according to their performance (best, middle, low) and selecting representatives for further characterisation in fedbatch processes is a way of dealing with the high number of clones and simultaneously not losing those with intrinsic variation before testing them in bioreactors (Mellitzer et al, 2012).…”

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

283

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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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

283

268

View full text Add to dashboard Cite

show abstract

“…The optimization and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in post-genomic science [11]. Herein, we recommend six ways to elevate the expression level of a recombinant protein.…”

Section: Discussionmentioning

confidence: 98%

High-Level Expression, Purification, Characterization and Structural Prediction of a Snake Venom Metalloproteinase Inhibitor in Pichia pastoris

Shi

et al. 2012

Protein J

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Snake venom metalloproteinase inhibitor BJ46a is from the serum of the venomous snake Bothrops jararaca. It has been proven to possess the capacity to inhibit matrix metalloproteinases (MMPs), likely based on its structural similarity to MMPs. This report describes the successful expression, purification, and characterization of the recombinant protein BJ46a in Pichia pastoris. Purified recombinant protein BJ46a was found to inhibit MMPs. Structural modeling was completed and should provide the foundation for further functional research. To our knowledge, this is the first report on the large scale expression of BJ46a, and it provides promise as a method for generation of BJ46a and investigation of its potential use as a new drug for treatment of antitumor invasion and metastasis.

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“…Another possibility for low activity and productivity of recombinant a-agarase in Pichia may be protein misfolding and protein degradation. In this case, the activity and productivity of a-agarase will be increased by introduction of molecular chaperones and chaperonins as protein folding and holding machines (Kwon et al 2002;Shin et al 2010) and optimization of culture condition (Holmes et al 2009;Routledge et al 2011). …”

Section: Determination Of the A-agarase Protein By Sds-pagementioning

confidence: 97%

Construction of an expression system for the secretory production of recombinant α-agarase in yeast

et al. 2012

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α-Agarase hydrolyzes the α-1,3 linkage of agarose yielding agaro-oligosaccharides. It is less well characterized than β-agarase. AgaA gene (2.3 kb ORF), encoding the α-agarase from Thalassomonas JAMB A33, was subcloned into both a constitutive and an inducible expression vector. Both the constructed plasmids, pVT-AgaA (ADH1 promoter) and pYInu-AgaA (GAL10 promoter), were transformed into Saccharomyces cerevisiae SEY2102 and FY833 and pPIC9-AgaA harboring the AOX1 promoter was transformed into Pichia pastoris GS115. The recombinant α-agarases were over-expressed with activities from 0.3 to 1.6 unit/ml, the highest being in the SEY2102/pYInu-AgaA transformant. Most of the recombinant α-agarase was extracellular because each plasmid possesses a signal sequence for the secretory production of α-agarase. In contrast, the Pichia host-vector expression system was unsuitable for the production of recombinant α-agarase. This is the first report of recombinant production of α-agarase in yeast for industrial use.

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Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

Cited by 67 publications

References 29 publications

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Cultivation strategies to enhance productivity of Pichia pastoris: A review

High-Level Expression, Purification, Characterization and Structural Prediction of a Snake Venom Metalloproteinase Inhibitor in Pichia pastoris

Construction of an expression system for the secretory production of recombinant α-agarase in yeast

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