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Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation

The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for hepari...

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Autores principales: Garcia-Pardo, Javier, Montané, Sergi, Avilés, Francesc Xavier, Tanco, Sebastian, Lorenzo, Julia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9609459/
https://www.ncbi.nlm.nih.gov/pubmed/36297573
http://dx.doi.org/10.3390/pharmaceutics14102138
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author Garcia-Pardo, Javier
Montané, Sergi
Avilés, Francesc Xavier
Tanco, Sebastian
Lorenzo, Julia
author_facet Garcia-Pardo, Javier
Montané, Sergi
Avilés, Francesc Xavier
Tanco, Sebastian
Lorenzo, Julia
author_sort Garcia-Pardo, Javier
collection PubMed
description The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies.
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spelling pubmed-96094592022-10-28 Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation Garcia-Pardo, Javier Montané, Sergi Avilés, Francesc Xavier Tanco, Sebastian Lorenzo, Julia Pharmaceutics Article The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies. MDPI 2022-10-08 /pmc/articles/PMC9609459/ /pubmed/36297573 http://dx.doi.org/10.3390/pharmaceutics14102138 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Garcia-Pardo, Javier
Montané, Sergi
Avilés, Francesc Xavier
Tanco, Sebastian
Lorenzo, Julia
Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation
title Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation
title_full Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation
title_fullStr Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation
title_full_unstemmed Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation
title_short Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation
title_sort enhanced production of ecm proteins for pharmaceutical applications using mammalian cells and sodium heparin supplementation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9609459/
https://www.ncbi.nlm.nih.gov/pubmed/36297573
http://dx.doi.org/10.3390/pharmaceutics14102138
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