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Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel (34)S labeling procedure

BACKGROUND: The budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intr...

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Autores principales: Pfeffer, Martin, Maurer, Michael, Köllensperger, Gunda, Hann, Stephan, Graf, Alexandra B, Mattanovich, Diethard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147017/
https://www.ncbi.nlm.nih.gov/pubmed/21703020
http://dx.doi.org/10.1186/1475-2859-10-47
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author Pfeffer, Martin
Maurer, Michael
Köllensperger, Gunda
Hann, Stephan
Graf, Alexandra B
Mattanovich, Diethard
author_facet Pfeffer, Martin
Maurer, Michael
Köllensperger, Gunda
Hann, Stephan
Graf, Alexandra B
Mattanovich, Diethard
author_sort Pfeffer, Martin
collection PubMed
description BACKGROUND: The budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask) and carbon-limited growth (bioreactor) conditions, they should be determined under "production like" conditions. Thus labeling procedures must be compatible with minimal production media and the usage of bioreactors. The inorganic and non-radioactive (34)S labeled sodium sulfate meets both demands. RESULTS: We used a novel labeling method with the stable sulfur isotope (34)S, administered as sodium sulfate, which is performed during chemostat culivations. The intra- and extracellular sulfur 32 to 34 ratios of purified recombinant protein, the antibody fragment Fab3H6, are measured by HPLC-ICP-MS. The kinetic model described here is necessary to calculate the kinetic parameters from sulfur ratios of consecutive samples as well as for sensitivity analysis. From the total amount of protein produced intracellularly (143.1 μg g(-1 )h(-1 )protein per yeast dry mass and time) about 58% are degraded within the cell, 35% are secreted to the exterior and 7% are inherited to the daughter cells. CONCLUSIONS: A novel (34)S labeling procedure that enables in vivo quantification of intracellular fluxes of recombinant protein under "production like" conditions is described. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most promising approaches for strain improvement towards increased secretion.
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spelling pubmed-31470172011-08-01 Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel (34)S labeling procedure Pfeffer, Martin Maurer, Michael Köllensperger, Gunda Hann, Stephan Graf, Alexandra B Mattanovich, Diethard Microb Cell Fact Research BACKGROUND: The budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask) and carbon-limited growth (bioreactor) conditions, they should be determined under "production like" conditions. Thus labeling procedures must be compatible with minimal production media and the usage of bioreactors. The inorganic and non-radioactive (34)S labeled sodium sulfate meets both demands. RESULTS: We used a novel labeling method with the stable sulfur isotope (34)S, administered as sodium sulfate, which is performed during chemostat culivations. The intra- and extracellular sulfur 32 to 34 ratios of purified recombinant protein, the antibody fragment Fab3H6, are measured by HPLC-ICP-MS. The kinetic model described here is necessary to calculate the kinetic parameters from sulfur ratios of consecutive samples as well as for sensitivity analysis. From the total amount of protein produced intracellularly (143.1 μg g(-1 )h(-1 )protein per yeast dry mass and time) about 58% are degraded within the cell, 35% are secreted to the exterior and 7% are inherited to the daughter cells. CONCLUSIONS: A novel (34)S labeling procedure that enables in vivo quantification of intracellular fluxes of recombinant protein under "production like" conditions is described. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most promising approaches for strain improvement towards increased secretion. BioMed Central 2011-06-26 /pmc/articles/PMC3147017/ /pubmed/21703020 http://dx.doi.org/10.1186/1475-2859-10-47 Text en Copyright ©2011 Pfeffer et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Pfeffer, Martin
Maurer, Michael
Köllensperger, Gunda
Hann, Stephan
Graf, Alexandra B
Mattanovich, Diethard
Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel (34)S labeling procedure
title Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel (34)S labeling procedure
title_full Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel (34)S labeling procedure
title_fullStr Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel (34)S labeling procedure
title_full_unstemmed Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel (34)S labeling procedure
title_short Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel (34)S labeling procedure
title_sort modeling and measuring intracellular fluxes of secreted recombinant protein in pichia pastoris with a novel (34)s labeling procedure
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147017/
https://www.ncbi.nlm.nih.gov/pubmed/21703020
http://dx.doi.org/10.1186/1475-2859-10-47
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