University of Oulu

Albert Galera-Prat, Juho Alaviuhkola, Heli I Alanen, Lari Lehtiö, Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity, Protein Engineering, Design and Selection, Volume 35, 2022, gzac006,

Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity

Saved in:
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:
Language: English
Published: Oxford University Press, 2022
Publish Date: 2023-02-02


Human 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

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
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
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 (, which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.