A journey down to hell: new thermostable protein-tags for biotechnology at high temperatures.

Published on Jan 1, 2020in Extremophiles2.046
· DOI :10.1007/S00792-019-01134-3
Rosanna Mattossovich2
Estimated H-index: 2
Rosa Merlo1
Estimated H-index: 1
+ 5 AuthorsGiuseppe Perugino18
Estimated H-index: 18
The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag® and the Halotag®. These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H5) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag® for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H5, proposing them as alternatives for in vivo studies in these extreme model organisms.
  • References (61)
  • Citations (1)
📖 Papers frequently viewed together
8 Citations
3 Citations
5 Authors (Gianfranco Peluso, ..., Mosè Rossi)
78% of Scinapse members use related papers. After signing in, all features are FREE.
#1Mario Serpe (National Research Council)H-Index: 5
#2Chiara Forenza (National Research Council)H-Index: 1
Last. Anna Valenti (National Research Council)H-Index: 11
view all 7 authors...
DNA alkylguanine DNA alkyltransferases (AGTs) are evolutionary conserved proteins that repair alkylation damage in DNA, counteracting the effects of agents inducing such lesions. Over the last years AGTs have raised considerable interest for both the peculiarity of their molecular mechanism and their relevance in cancer biology. AGT knock out mice show increased tumour incidence in response to alkylating agents, and over-expression of the human AGT protein in cancer cells is frequently associate...
1 CitationsSource
#1Kosuke Aoki (Nagoya University)H-Index: 4
#2Atsushi Natsume (Nagoya University)H-Index: 35
Adult diffuse gliomas form a heterogeneous group of tumors of the central nervous system that vary greatly in histology and prognosis. A significant advance during the last decade has been the identification of a set of genetic lesions that correlate well with histology and clinical outcome in diffuse gliomas. Most characteristic driver mutations consist of isocitrate dehydrogenase 1 (IDH1) and IDH2, and H3 histone family member 3A, which are strongly associated with DNA and histone methylation ...
1 CitationsSource
#1Giada Lo Gullo (Sapienza University of Rome)H-Index: 1
#2Rosanna Mattossovich (National Research Council)H-Index: 2
Last. Dario Benelli (Sapienza University of Rome)H-Index: 12
view all 6 authors...
A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mR...
2 CitationsSource
#1Sonia Del Prete (National Research Council)H-Index: 23
#2Rosa Merlo (National Research Council)H-Index: 1
Last. Clemente Capasso (National Research Council)H-Index: 40
view all 9 authors...
AbstractCarbonic anhydrases (CAs, EC are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with ...
1 CitationsSource
#1Rosa Merlo (National Research Council)H-Index: 1
#2Sonia Del Prete (National Research Council)H-Index: 23
Last. Giuseppe Perugino (National Research Council)H-Index: 18
view all 8 authors...
AbstractThe use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of prote...
2 CitationsSource
#1Guohui Sun (Beijing University of Technology)H-Index: 6
#2Lijiao Zhao (Beijing University of Technology)H-Index: 7
Last. Yongzhen Peng (Beijing University of Technology)H-Index: 3
view all 4 authors...
The DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), can confer resistance to guanine O6-alkylating agents. Therefore, inhibition of resistant MGMT protein is a practical approach to increase the anticancer effects of such alkylating agents. Numerous small molecule inhibitors were synthesized and exhibited potential MGMT inhibitory activities. Although they were nontoxic alone, they also inhibited MGMT in normal tissues, thereby enhancing the side effects of chemotherapy. There...
7 CitationsSource
O6-DNA-alkyl-guanine-DNA-alkyl-transferases (OGTs) are evolutionarily conserved, unique proteins that repair alkylation lesions in DNA in a single step reaction. Alkylating agents are environmental pollutants as well as by-products of cellular reactions, but are also very effective chemotherapeutic drugs. OGTs are major players in counteracting the effects of such agents, thus their action in turn affects genome integrity, survival of organisms under challenging conditions and response to chemot...
5 CitationsSource
#1Servé W. M. Kengen (WUR: Wageningen University and Research Centre)H-Index: 34
Pyrococcus furiosus has come of age. In 1986 the first publication on a remarkable microorganism, Pyrococcus furiosus, appeared. Now, 30 years later it is still "the fast and the furious".
3 CitationsSource
#1Valeria VisoneH-Index: 4
#2Wenyuan HanH-Index: 1
Last. Maria CiaramellaH-Index: 23
view all 8 authors...
: Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high t...
9 CitationsSource
#1Nirakar Pradhan (University of Cassino)H-Index: 2
#2Laura Dipasquale (National Research Council)H-Index: 13
Last. Angelo Fontana (National Research Council)H-Index: 16
view all 7 authors...
Abstract Thermotoga neapolitana is a hyperthermophilic eubacterium that produces hydrogen by sugar fermentation. A lab strain of T. neapolitana DSMZ 4359T maintained in a CO2-enriched atmosphere showed a stable increase of lactic acid production under capnophilic lactic fermentation (CLF) conditions. The genotypic comparison between the putative mutant (TN-CMut) and the original strain DSMZ 4359T (WT4359) revealed 88.1 (±2.4)% DNA homology. RiboPrint® and MALDI-TOF mass analyses support a geneti...
10 CitationsSource
Cited By1
The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-posi...