New Biotech tool from Hot Sources: Thermostable self-labeling protein-tags near to the boiling water

Abstract In the past decade, a powerful biotechnological tool for specific in vivo and in vitro labeling of proteins of interest, the SNAP-tag technology, has been proposed as a valid alternative to the classic protein-tags. This self-labeling protein-tag is an engineered variant of the human O6-alkylguanine-DNA-alkyltransferases (hMGMT) enzyme, which specifically reacts with benzyl-guanine (BG) derivatives, maintaining a part of these substrates in the catalytic site, with a one-step reaction. However, given the mesophilic nature of this protein-tag, the general use of this new approach is restricted to mesophilic model systems and mild reaction conditions. To overcome this limitation, it is necessary the exploitation of homologous activities from extremophilic sources, to apply this technology also to (hyper)thermophilic organisms and harsher reaction conditions. To this purpose, it has been first characterized an archaeal alkylguanine-DNA-alkyltransferases (AGT or OGT) from Saccharolobus solfataricus (SsOGT), which presented an exceptional in vitro stability at high temperatures, physical and chemical denaturing agents, making it suitable not only for the in vivo heterologous expression in Escherichia coli, but also in the thermophilic Thermus thermophilus and Sulfolobus islandicus organisms. Furthermore, the growing demand to apply this biotechnological tool to extreme conditions led to look for new protein-tags from hot sources. Recently, new AGTs from Thermotoga neapolitana (TnOGT) and Pyrococcus furiosus (PfuOGT) resulted different from SsOGT in terms of activity and stability, proposing them as potential tool for the in vivo analysis and function of enzymes of interest in these hyperthermophilic model systems.