A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA- alkyl-transferase by protein engineering.
In the last decade, a powerful biotechnological tool for the in vivo and in vitro specific labeling of proteins (SNAP-tag™ technology) was proposed as a valid alternative to classical protein-tags (green fluorescent proteins, GFPs). This was made possible by the discovery of the irreversible reaction of the human alkylguanine-DNA-alkyl-transferase (hAGT) in the presence of benzyl-guanine derivatives. However, the mild reaction conditions and the general instability of the mesophilic SNAP-tag™ make this new approach not fully applicable to (hyper-)thermophilic and, in general, extremophilic organisms. Here, we introduce an engineered variant of the thermostable alkylguanine-DNA-alkyl-transferase from the Archaea Sulfolobus solfataricus (SsOGT-H5), which displays a catalytic efficiency comparable to the SNAP-tag™ protein, but showing high intrinsic stability typical of proteins from this organism. The successful heterologous expression obtained in a thermophilic model organism makes SsOGT-H5 a valid candidate as protein-tag for organisms living in extreme environments.
Institute of Biosciences and BioResources, National Council of Research of Italy, Via P. Castellino 111, 80131 Naples, Italy
Antonella
A
Vettone
Mario
M
Serpe
Aurelio
A
Hidalgo
José
J
Berenguer
Giovanni
G
del Monaco
Anna
A
Valenti
Mosé
M
Rossi
Maria
M
Ciaramella
Giuseppe
G
Perugino
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase