In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

Autor: Qunxin She, Maria Ciaramella, Valeria Visone, Giuseppe Perugino, Mosè Rossi, Wenyuan Han, Giovanni del Monaco, Anna Valenti
Přispěvatelé: Visone, Valeria, Han, Wenyuan, Perugino, Giuseppe, Del Monaco, Giovanni, She, Qunxin, Rossi, Mosè, Valenti, Anna, Ciaramella, Maria
Jazyk: angličtina
Rok vydání: 2017
Předmět:
0301 basic medicine
Hot Temperature
Molecular biology
Protein Extraction
ved/biology.organism_classification_rank.species
Protein Expression
lcsh:Medicine
Protein tag
DNA annealing
Biochemistry
Fluorescence Microscopy
CRISPR
lcsh:Science
topoisomerase
Extraction Techniques
Microscopy
Multidisciplinary
Physics
Sulfolobus solfataricus
Light Microscopy
Sulfolobu
Small molecule
Sulfolobus
Physical sciences
Nucleic acids
In Vivo Imaging
DNA Topoisomerases
Type I
Nucleic acid thermodynamics
DNA supercoil
Research Article
Imaging Techniques
Archaeal Proteins
Annealing (genetics)
dna repair
Biophysics
Biology
DNA construction
03 medical and health sciences
Extremophiles
Archaeal Protein
Fluorescence Imaging
Gene Expression and Vector Techniques
Gene
Molecular Biology Assays and Analysis Techniques
Biology and life sciences
ved/biology
lcsh:R
Ecology and Environmental Sciences
biology.organism_classification
Archaea
In vitro
Research and analysis methods
030104 developmental biology
Molecular biology techniques
Plasmid Construction
lcsh:Q
Zdroj: Visone, V, Han, W, Perugino, G, del Monaco, G, She, Q, Rossi, M, Valenti, A & Ciaramella, M 2017, ' In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag ', PLoS ONE, vol. 12, no. 10, e0185791 . https://doi.org/10.1371/journal.pone.0185791
PLoS ONE
PloS one (2017). doi:10.1371/journal.pone.0185791
info:cnr-pdr/source/autori:Valeria Visone, Wenyuan Han, Giuseppe Perugino, Giovanni del Monaco, Qunxin She, Mosè Rossi, Anna Valenti*, Maria Ciaramella*/titolo:In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag/doi:10.1371%2Fjournal.pone.0185791/rivista:PloS one/anno:2017/pagina_da:/pagina_a:/intervallo_pagine:/volume
PLoS ONE, Vol 12, Iss 10, p e0185791 (2017)
DOI: 10.1371/journal.pone.0185791
Popis: 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 temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we con- structed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (?ogt). Introduction of a plasmid- borne H5 gene in this strain led to production of a functional H5 protein, which was success- fully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in ?ogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topo- isomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.
Databáze: OpenAIRE
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