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Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

ABSTRACT: The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constru...

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Autores principales: Moya-Torres, Aniel, Gupta, Monika, Heide, Fabian, Krahn, Natalie, Legare, Scott, Nikodemus, Denise, Imhof, Thomas, Meier, Markus, Koch, Manuel, Stetefeld, Jörg
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390410/
https://www.ncbi.nlm.nih.gov/pubmed/34342709
http://dx.doi.org/10.1007/s00253-021-11438-0
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author Moya-Torres, Aniel
Gupta, Monika
Heide, Fabian
Krahn, Natalie
Legare, Scott
Nikodemus, Denise
Imhof, Thomas
Meier, Markus
Koch, Manuel
Stetefeld, Jörg
author_facet Moya-Torres, Aniel
Gupta, Monika
Heide, Fabian
Krahn, Natalie
Legare, Scott
Nikodemus, Denise
Imhof, Thomas
Meier, Markus
Koch, Manuel
Stetefeld, Jörg
author_sort Moya-Torres, Aniel
collection PubMed
description ABSTRACT: The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-021-11438-0.
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spelling pubmed-83904102021-09-14 Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor Moya-Torres, Aniel Gupta, Monika Heide, Fabian Krahn, Natalie Legare, Scott Nikodemus, Denise Imhof, Thomas Meier, Markus Koch, Manuel Stetefeld, Jörg Appl Microbiol Biotechnol Methods and Protocols ABSTRACT: The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-021-11438-0. Springer Berlin Heidelberg 2021-08-03 2021 /pmc/articles/PMC8390410/ /pubmed/34342709 http://dx.doi.org/10.1007/s00253-021-11438-0 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Methods and Protocols
Moya-Torres, Aniel
Gupta, Monika
Heide, Fabian
Krahn, Natalie
Legare, Scott
Nikodemus, Denise
Imhof, Thomas
Meier, Markus
Koch, Manuel
Stetefeld, Jörg
Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor
title Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor
title_full Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor
title_fullStr Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor
title_full_unstemmed Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor
title_short Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor
title_sort homogenous overexpression of the extracellular matrix protein netrin-1 in a hollow fiber bioreactor
topic Methods and Protocols
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390410/
https://www.ncbi.nlm.nih.gov/pubmed/34342709
http://dx.doi.org/10.1007/s00253-021-11438-0
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