Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity |
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Author: | Galera-Prat, Albert1; Alaviuhkola, Juho1; Alanen, Heli I.1; |
Organizations: |
1Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, Aapistie 7B, 90220 Oulu, University of Oulu, Finland |
Format: | article |
Version: | published version |
Access: | open |
Online Access: | PDF Full Text (PDF, 1.4 MB) |
Persistent link: | http://urn.fi/urn:nbn:fi-fe2023020225592 |
Language: | English |
Published: |
Oxford University Press,
2022
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Publish Date: | 2023-02-02 |
Description: |
AbstractHuman mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover inhibitors have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different enzymes. We engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their activity using a homogeneous NAD⁺ conversion assay, facilitating its automation to lower the assay volume and costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity. see all
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Series: |
Protein engineering design & selection |
ISSN: | 1741-0126 |
ISSN-E: | 1741-0134 |
ISSN-L: | 1741-0126 |
Volume: | 35 |
Article number: | gzac006 |
DOI: | 10.1093/protein/gzac006 |
OADOI: | https://oadoi.org/10.1093/protein/gzac006 |
Type of Publication: |
A1 Journal article – refereed |
Field of Science: |
1182 Biochemistry, cell and molecular biology |
Subjects: | |
Funding: |
The work was supported by the Sigrid Jusélius and Jane and Aatos Erkko foundations (LL). |
Copyright information: |
© The Author(s) 2022. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
https://creativecommons.org/licenses/by/4.0/ |