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

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 mea...

Full description

Bibliographic Details
Main Authors: Valeria Visone, Wenyuan Han, Giuseppe Perugino, Giovanni Del Monaco, Qunxin She, Mosè Rossi, Anna Valenti, Maria Ciaramella
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2017-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC5626487?pdf=render
_version_ 1828768010295312384
author Valeria Visone
Wenyuan Han
Giuseppe Perugino
Giovanni Del Monaco
Qunxin She
Mosè Rossi
Anna Valenti
Maria Ciaramella
author_facet Valeria Visone
Wenyuan Han
Giuseppe Perugino
Giovanni Del Monaco
Qunxin She
Mosè Rossi
Anna Valenti
Maria Ciaramella
author_sort Valeria Visone
collection DOAJ
description 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 constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase 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.
first_indexed 2024-12-11T07:38:58Z
format Article
id doaj.art-e404c1ac4bf44ab69be0098405853b77
institution Directory Open Access Journal
issn 1932-6203
language English
last_indexed 2024-12-11T07:38:58Z
publishDate 2017-01-01
publisher Public Library of Science (PLoS)
record_format Article
series PLoS ONE
spelling doaj.art-e404c1ac4bf44ab69be0098405853b772022-12-22T01:15:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-011210e018579110.1371/journal.pone.0185791In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.Valeria VisoneWenyuan HanGiuseppe PeruginoGiovanni Del MonacoQunxin SheMosè RossiAnna ValentiMaria CiaramellaProtein 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 constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase 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.http://europepmc.org/articles/PMC5626487?pdf=render
spellingShingle Valeria Visone
Wenyuan Han
Giuseppe Perugino
Giovanni Del Monaco
Qunxin She
Mosè Rossi
Anna Valenti
Maria Ciaramella
In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.
PLoS ONE
title In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.
title_full In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.
title_fullStr In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.
title_full_unstemmed In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.
title_short In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.
title_sort in vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag
url http://europepmc.org/articles/PMC5626487?pdf=render
work_keys_str_mv AT valeriavisone invivoandinvitroproteinimaginginthermophilicarchaeabyexploitinganovelproteintag
AT wenyuanhan invivoandinvitroproteinimaginginthermophilicarchaeabyexploitinganovelproteintag
AT giuseppeperugino invivoandinvitroproteinimaginginthermophilicarchaeabyexploitinganovelproteintag
AT giovannidelmonaco invivoandinvitroproteinimaginginthermophilicarchaeabyexploitinganovelproteintag
AT qunxinshe invivoandinvitroproteinimaginginthermophilicarchaeabyexploitinganovelproteintag
AT moserossi invivoandinvitroproteinimaginginthermophilicarchaeabyexploitinganovelproteintag
AT annavalenti invivoandinvitroproteinimaginginthermophilicarchaeabyexploitinganovelproteintag
AT mariaciaramella invivoandinvitroproteinimaginginthermophilicarchaeabyexploitinganovelproteintag