Recombinant Protein Production with Pichia pastoris in Continuous Fermentation – Kinetic Analysis of Growth and Product Formation

Curvers, Simon; Linnemann, Jorg; Klauser, Thomas; Wandrey, Christian; Takors, Ralf

doi:10.1002/1618-2863(20020806)2:8<229::aid-elsc229>3.0.co;2-9

Cited by 40 publications

(48 citation statements)

References 11 publications

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“…Nevertheless, Ohi et al (1998) reported that after serial cultivation of a P. pastoris strain expressing human serum albumin (two copies) for 83 generations, 0.01% of cells were found to lose the foreign gene. Also, Curvers et al (2002) reported that in continuous cultivation of a recombinant P. pastoris strain expressing recombinant human chymotrypsinogen B (single copy), yeast cells in the bioreactor soon lost the ability to express the protein due to the instability of P. pastoris when the residual methanol exceeded 4 g l -1 .…”

Section: Discussionmentioning

confidence: 99%

A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains

et al. 2009

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A battery of Pichia pastoris transformants, G1, G6, A2, A3, C3, carrying 1, 6, 12, 18 and 29 copies of porcine insulin precursor (PIP) gene, were employed to investigate the genetic stability of these multi-copy P. pastoris strains. Both G6 and C3 maintained their original copy numbers in serial culture without methanol induction for 35 generations. With methanol as an inducer and carbon source, G1 and G6 remained stable but the average copy numbers (ACNs) of PIP gene in A2, A3, C3 were decreased to 10, 10 and 15 copies, respectively, after 96 h of induction in shake-flask culture. A PIP copy number distribution analysis of fermentation samples of C3 indicated that the majority of yeast cells have partially or completely lost their PIP genes. In 5-l fermentor culture, the ACNs of PIP gene in A2, A3, C3 were also decreased to 10, 15, 21 copies, respectively, after 72 h of methanol induction.

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Section: Discussionmentioning

confidence: 99%

A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains

et al. 2009

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“…In this study, 100% methanol was used to a final concentration of 0.3-0.5% every 24 h, and the highest expression was made in 0.5% of methanol. Curvers and colleagues' results showed that when methanol concentration increases more than a critical point, a selective pressure will be imposed against product formation leading to the enrichment of nonproducing mutants [36].…”

Section: Discussionmentioning

confidence: 99%

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in Pichia pastoris

Majidzadeh‐A

Khalaj

Davami

et al. 2010

Appl Biochem Biotechnol

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Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS-PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.

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“…Scenarios G1-G10 have been extracted from P. pastoris cultures producing recombinant human chymotrypsinogen B [36]. Scenario H1 corresponds to the continuous fermentation of a P. pastoris strain for the extracellular production of a recombinant ovine interferon protein [37].…”

Section: P Pastoris Experimental Data Setmentioning

confidence: 99%

MCR-ALS on metabolic networks: Obtaining more meaningful pathways

Folch-Fortuny

Tortajada

Prats-Montalbán

et al. 2015

Chemometrics and Intelligent Laboratory Systems

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With the aim of understanding the flux distributions across a metabolic network, i.e. within living cells, Principal Component Analysis (PCA) has been proposed to obtain a set of orthogonal components (pathways) capturing most of the variance in the flux data. The problems with this method are (i) that no additional information can be included in the model, and (ii) that orthogonality imposes a hard constraint, not always reasonably. To overcome these drawbacks, here we propose to use a more flexible approach such as Multivariate Curve Resolution -Alternating Least Squares (MCR-ALS) to obtain this set of biological pathways through the network. By using this method, different constraints can be included in the model, and the same source of variability can be present in different pathways, which is reasonable from a biological standpoint. This work follows a methodology developed for Pichia pastoris cultures grown on different carbon sources, lately presented in [González Martínez et al. 2014].In this paper a different grey modelling approach, which aims to incorporate a priori knowledge through constraints on the modelling algorithms, is applied to the same case of study. The results of both models are compared to show their strengths and weaknesses.

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Recombinant Protein Production with Pichia pastoris in Continuous Fermentation – Kinetic Analysis of Growth and Product Formation

Cited by 40 publications

References 11 publications

A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains

A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in Pichia pastoris

MCR-ALS on metabolic networks: Obtaining more meaningful pathways

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