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PROFICS: A bacterial selection system for directed evolution of proteases
Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Car...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8446807/ https://www.ncbi.nlm.nih.gov/pubmed/34418435 http://dx.doi.org/10.1016/j.jbc.2021.101095 |
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author | Kröß, Christina Engele, Petra Sprenger, Bernhard Fischer, Andreas Lingg, Nico Baier, Magdalena Öhlknecht, Christoph Lier, Bettina Oostenbrink, Chris Cserjan-Puschmann, Monika Striedner, Gerald Jungbauer, Alois Schneider, Rainer |
author_facet | Kröß, Christina Engele, Petra Sprenger, Bernhard Fischer, Andreas Lingg, Nico Baier, Magdalena Öhlknecht, Christoph Lier, Bettina Oostenbrink, Chris Cserjan-Puschmann, Monika Striedner, Gerald Jungbauer, Alois Schneider, Rainer |
author_sort | Kröß, Christina |
collection | PubMed |
description | Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner. |
format | Online Article Text |
id | pubmed-8446807 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-84468072021-09-22 PROFICS: A bacterial selection system for directed evolution of proteases Kröß, Christina Engele, Petra Sprenger, Bernhard Fischer, Andreas Lingg, Nico Baier, Magdalena Öhlknecht, Christoph Lier, Bettina Oostenbrink, Chris Cserjan-Puschmann, Monika Striedner, Gerald Jungbauer, Alois Schneider, Rainer J Biol Chem Research Article Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner. American Society for Biochemistry and Molecular Biology 2021-08-19 /pmc/articles/PMC8446807/ /pubmed/34418435 http://dx.doi.org/10.1016/j.jbc.2021.101095 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Kröß, Christina Engele, Petra Sprenger, Bernhard Fischer, Andreas Lingg, Nico Baier, Magdalena Öhlknecht, Christoph Lier, Bettina Oostenbrink, Chris Cserjan-Puschmann, Monika Striedner, Gerald Jungbauer, Alois Schneider, Rainer PROFICS: A bacterial selection system for directed evolution of proteases |
title | PROFICS: A bacterial selection system for directed evolution of proteases |
title_full | PROFICS: A bacterial selection system for directed evolution of proteases |
title_fullStr | PROFICS: A bacterial selection system for directed evolution of proteases |
title_full_unstemmed | PROFICS: A bacterial selection system for directed evolution of proteases |
title_short | PROFICS: A bacterial selection system for directed evolution of proteases |
title_sort | profics: a bacterial selection system for directed evolution of proteases |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8446807/ https://www.ncbi.nlm.nih.gov/pubmed/34418435 http://dx.doi.org/10.1016/j.jbc.2021.101095 |
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