O6-alkylguanine-DNA Alkyltransferase (AGT): when Nature meets Biotechnology

Rosa Merlo 
IBBR - UOS Napoli - Italy

February 17, 2021 (11:30-12:30)
Webinar Link: (not available yet)

Abstract: O 6 -alkylguanine-DNA-alkyltransferases (AGT or OGT, EC 2.1.1.63) are small enzymes involved in the removal of alkyl groups from the damaged DNA, through an irreversible single-step reaction . This mechanism led to the development of useful biotechnological tools for the specific labelling of proteins , such as the so-called SNAP-tag® technology from New England Biolabs (https://international.neb.com/tools-and-resources/feature-articles/snap-tag-technologies-noveltools-to-study-protein-function). SNAP-tag® allows the indirect covalent labelling with a desired chemical group L (previously conjugated to its substrate) of a protein of interest (POI) , without affecting the activity and stability of the latter. Since the SNAP-tag® is based on a peculiar enzyme and on particular substrates, we improved and expanded this technology by operating on both: i) synthesis and purification procedures of each SNAP-substrate are necessary steps, thus increasing time and costs, with a risk to lower the catalytic activity of this protein-tag. To get over this issue, we propose a revision of this technique, by developing a chemo-enzymatic approach with a selected azide-based SNAP-substrate (BGSN); ii) the growing demand to apply this type of protein-tag to extreme conditions and in (hyper)thermophilic organisms, led to look for AGTs from hot sources, because SNAP-tag® is a mesophilic enzyme. Therefore, from the archaeon Saccharolobus solfataricus (SsOGT) and from Pyrococcus furiosus (PfuOGT) we identified, characterized and then engineered new AGTs, leading to the first (hyper)thermo-SNAP-tags known so far, opening new perspectives and further widening the applications of the SNAP-technology

Author's Info: https://ibbr.cnr.it/ibbr/info/people/rosa-merlo

Link to video: https://youtu.be/Kk1_ht6AWpA