Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology

Environmental Microbiology
Felipe CavaJosé Berenguer

Abstract

Superfolder GFP (sGFP) is a variant of the Green Fluorescent Protein that folds efficiently when fused to poorly folded proteins. In this study, we show that sGFP, but not enhanced GFP, is functional in vivo at 70 degrees C in the extreme thermophile Thermus thermophilus (Tth); thus, permitting the use of sGFP as a localization tag in vivo. We created a suite of plasmids that allow the expression of carboxy-terminal sGFP fusion proteins in both Escherichia coli and Tth. In order to demonstrate the facility of sGFP as an in vivo localization tag in Tth, we tagged GroES (the small subunit of the bacterial GroES/GroEL chaperone), NarC (a membrane component of the nitrate respiration apparatus) and PhoA (a TAT-secreted periplasmic protein), and visualized the distribution of the sGFP fusion proteins using confocal microscopy. Fusions to NarC and PhoA produced enzymatically active proteins that complemented both the narC and the phoA strains respectively. Observation of the distribution of the GroES-sGFP protein by confocal microscopy revealed a homogeneous fluorescence in the cells, which is in full agreement with the cytoplasmic nature of GroES, whereas the NarC-sGFP protein was localized to the membrane. Finally, a combination of...Continue Reading

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Citations

May 8, 2008·Applied Microbiology and Biotechnology·Margarita DíazRamón Santamaría
Jan 22, 2009·Extremophiles : Life Under Extreme Conditions·Felipe CavaJosé Berenguer
Nov 22, 2011·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Craig Don PaulMark Prescott
Feb 18, 2014·Nature·Daan C SwartsJohn van der Oost
Apr 25, 2012·Extremophiles : Life Under Extreme Conditions·Federico AcostaJosé Berenguer
Jan 31, 2012·Journal of Biotechnology·Birgit KlinkertFranz Narberhaus
Aug 16, 2011·Journal of Biotechnology·Juan M BolivarFernando López-Gallego
Oct 27, 2015·Extremophiles : Life Under Extreme Conditions·Antonella VettoneGiuseppe Perugino
Jun 28, 2013·Molecular Microbiology·Annika CimdinsFranz Narberhaus
Sep 10, 2013·Protein Expression and Purification·Jie FangJun Fan
Jan 1, 2012·Computational and Structural Biotechnology Journal·Thorben Dammeyer, Philip Tinnefeld
Jun 21, 2014·Extremophiles : Life Under Extreme Conditions·Jennifer F CarrAlbert E Dahlberg
Jun 23, 2016·ACS Synthetic Biology·Benjamin ReeveTom Ellis
Apr 23, 2015·PloS One·Daan C SwartsJohn van der Oost
May 14, 2017·Photochemistry and Photobiology·Marcus WingenThomas Drepper
Dec 30, 2015·Applied and Environmental Microbiology·Naoto OhtaniMitsuhiro Itaya
Jul 26, 2017·Proceedings of the National Academy of Sciences of the United States of America·Julie M PelletierJohn J Harada
Jul 18, 2019·International Journal of Molecular Sciences·Jean-Denis Pedelacq, Stéphanie Cabantous
May 12, 2018·Microorganisms·Eivind B DrejerTrygve Brautaset

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