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Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization
Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760212/ https://www.ncbi.nlm.nih.gov/pubmed/33255244 http://dx.doi.org/10.3390/biom10121592 |
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author | Cserjan-Puschmann, Monika Lingg, Nico Engele, Petra Kröß, Christina Loibl, Julian Fischer, Andreas Bacher, Florian Frank, Anna-Carina Öhlknecht, Christoph Brocard, Cécile Oostenbrink, Chris Berkemeyer, Matthias Schneider, Rainer Striedner, Gerald Jungbauer, Alois |
author_facet | Cserjan-Puschmann, Monika Lingg, Nico Engele, Petra Kröß, Christina Loibl, Julian Fischer, Andreas Bacher, Florian Frank, Anna-Carina Öhlknecht, Christoph Brocard, Cécile Oostenbrink, Chris Berkemeyer, Matthias Schneider, Rainer Striedner, Gerald Jungbauer, Alois |
author_sort | Cserjan-Puschmann, Monika |
collection | PubMed |
description | Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at −20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process. |
format | Online Article Text |
id | pubmed-7760212 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-77602122020-12-26 Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization Cserjan-Puschmann, Monika Lingg, Nico Engele, Petra Kröß, Christina Loibl, Julian Fischer, Andreas Bacher, Florian Frank, Anna-Carina Öhlknecht, Christoph Brocard, Cécile Oostenbrink, Chris Berkemeyer, Matthias Schneider, Rainer Striedner, Gerald Jungbauer, Alois Biomolecules Article Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at −20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process. MDPI 2020-11-24 /pmc/articles/PMC7760212/ /pubmed/33255244 http://dx.doi.org/10.3390/biom10121592 Text en © 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Cserjan-Puschmann, Monika Lingg, Nico Engele, Petra Kröß, Christina Loibl, Julian Fischer, Andreas Bacher, Florian Frank, Anna-Carina Öhlknecht, Christoph Brocard, Cécile Oostenbrink, Chris Berkemeyer, Matthias Schneider, Rainer Striedner, Gerald Jungbauer, Alois Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization |
title | Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization |
title_full | Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization |
title_fullStr | Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization |
title_full_unstemmed | Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization |
title_short | Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization |
title_sort | production of circularly permuted caspase-2 for affinity fusion-tag removal: cloning, expression in escherichia coli, purification, and characterization |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760212/ https://www.ncbi.nlm.nih.gov/pubmed/33255244 http://dx.doi.org/10.3390/biom10121592 |
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