A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA- alkyl-transferase by protein engineering

Extremophiles. 2016 Jan;20(1):1-13. doi: 10.1007/s00792-015-0791-9.

Abstract

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.

MeSH terms

  • Alkyl and Aryl Transferases / chemistry
  • Alkyl and Aryl Transferases / genetics
  • Alkyl and Aryl Transferases / metabolism*
  • Amino Acid Sequence
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • DNA / metabolism
  • Enzyme Stability
  • Hot Temperature
  • Molecular Sequence Data
  • Protein Binding
  • Protein Engineering
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sulfolobus solfataricus / enzymology*
  • Sulfolobus solfataricus / genetics

Substances

  • Archaeal Proteins
  • Recombinant Fusion Proteins
  • DNA
  • Alkyl and Aryl Transferases
  • DNA alkyltransferase