Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics

Fujiki, Y.; Kumada, Yoichi; Kishimoto, Michimasa

doi:10.1016/j.jbiosc.2014.12.015

Cited by 3 publications

(1 citation statement)

References 21 publications

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“…One of the reasons for this is that the P. pastoris cell that expresses antimicrobial peptides is often unstable after methanol treatments such as autolysis. Thus, cell autolysis is one of the most noteworthy issues to be solved for the recombinant protein expression system [ 4 ]. Conversely, the extraction and purification of a recombinant protein may contribute to 80% or more of the total production cost for a gene expression system, and the fact that cells have to be lysed by drastic methods to be recovered can affect the product and the cost [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris

Wang

Sheng

et al. 2022

Molecules

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Autolysis is a common physiological process in eukaryotic cells that is often prevented or applied, especially in yeast expression systems. In this study, an antimicrobial peptide from chicken (AMP) was recombinantly expressed in the Pichia pastoris expression system, which induced a series of cellular autolysis phenotypes after methanol treatment, such as the aggregated, lysed, irregular, and enlarged cell morphology, while the cells expressing a recombinant aflatoxin-detoxifizyme (ADTZ) were not autolyzed. A comparative transcriptomic analysis showed that the transcriptomic profiles of cells derived from the autolysis and non-autolysis groups were well discriminated, suggesting that the mechanisms of autolysis were at the transcriptional level. A further differential expression gene (DEG) analysis showed that the DEGs from the two groups were involved mainly in autophagy, the MAPK signaling pathway, transcriptional factors, the central carbon metabolism, anti-stress functions, and so on. In the autolysis group, the cell activity was significantly reduced with the MAPK signaling pathway, the central carbon metabolism was down-regulated, and components of the cytoplasm-to-vacuole targeting (CVT) and mitophagy pathways were up-regulated, suggesting that the autophagy involved in the trafficking of intracellular molecules in the vacuole and mitochondrion contributed to autolysis, which was regulated by transcriptional factors and signal pathways at the transcriptional level. This study provides a theoretical basis for genetic modifications to prevent or utilize cell autolysis in the recombinant protein expression system.

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Section: Introductionmentioning

confidence: 99%

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris

Wang

Sheng

et al. 2022

Molecules

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Strategies for selection and identification of rabbit single-chain Fv antibodies as ligand in affinity chromatography

Kumada

Rakotondravao

Hasegawa

et al. 2022

Journal of Bioscience and Bioengineering

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Systems biotechnology for protein production in Pichia pastoris

Zahrl

Peña

Mattanovich

et al. 2017

FEMS Yeast Research

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The methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) is one of the most important production systems for heterologous proteins. After the first genome sequences were published in 2009, tremendous effort was made to establish systems-level analytical methods. Methylotrophic lifestyle was one of the most thoroughly investigated topics, studied at the levels of transcriptome, proteome and metabolic flux. Also the responses of P. pastoris to environmental stress conditions experienced during high cell density production processes were studied. Metabolomics and flux analysis revealed the plasticity of the cellular metabolism in its adaption to the production of foreign proteins and served as blueprints for subsequent cell engineering and/or process design. The transcriptional response elicited by overexpression of heterologous proteins seems to depend on the nature and complexity of the recombinant product. Based on these data, novel targets for strain engineering could be deduced from transcriptomics and proteomics data mining and effectively enhanced protein secretion. Transcriptional regulation data also served as a valuable resource to identify novel promoters with the desired regulatory characteristics. This review aims to provide a comprehensive overview of systems biology applications in P. pastoris ranging from increased understanding of cell physiology to improving recombinant protein production in this cell factory.

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Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics

Cited by 3 publications

References 21 publications

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris

Strategies for selection and identification of rabbit single-chain Fv antibodies as ligand in affinity chromatography

Systems biotechnology for protein production in Pichia pastoris

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