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Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the pr...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Vienna
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9117350/ https://www.ncbi.nlm.nih.gov/pubmed/34453584 http://dx.doi.org/10.1007/s00726-021-03004-9 |
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author | Sandberg, Magdalena Widgren Bunkenborg, Jakob Thyssen, Stine Villadsen, Martin Kofoed, Thomas |
author_facet | Sandberg, Magdalena Widgren Bunkenborg, Jakob Thyssen, Stine Villadsen, Martin Kofoed, Thomas |
author_sort | Sandberg, Magdalena Widgren |
collection | PubMed |
description | Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C(4)H(6)O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00726-021-03004-9. |
format | Online Article Text |
id | pubmed-9117350 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Springer Vienna |
record_format | MEDLINE/PubMed |
spelling | pubmed-91173502022-05-20 Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry Sandberg, Magdalena Widgren Bunkenborg, Jakob Thyssen, Stine Villadsen, Martin Kofoed, Thomas Amino Acids Original Article Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C(4)H(6)O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00726-021-03004-9. Springer Vienna 2021-08-28 2022 /pmc/articles/PMC9117350/ /pubmed/34453584 http://dx.doi.org/10.1007/s00726-021-03004-9 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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 | Original Article Sandberg, Magdalena Widgren Bunkenborg, Jakob Thyssen, Stine Villadsen, Martin Kofoed, Thomas Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry |
title | Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry |
title_full | Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry |
title_fullStr | Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry |
title_full_unstemmed | Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry |
title_short | Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry |
title_sort | characterization of a novel + 70 da modification in rhgm-csf expressed in e. coli using chemical assays in combination with mass spectrometry |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9117350/ https://www.ncbi.nlm.nih.gov/pubmed/34453584 http://dx.doi.org/10.1007/s00726-021-03004-9 |
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