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Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD

The human Fic domain-containing protein (FICD) is a type II endoplasmic reticulum (ER) membrane protein that is important for the maintenance of ER proteostasis. Structural and in vitro biochemical characterisation of FICD AMPylase and deAMPylase activity have been restricted to the soluble ER-lumin...

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Autores principales: Virolainen, Minttu S., Søltoft, Cecilie L., Pedersen, Per A., Ellgaard, Lars
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8910494/
https://www.ncbi.nlm.nih.gov/pubmed/35269596
http://dx.doi.org/10.3390/ijms23052458
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author Virolainen, Minttu S.
Søltoft, Cecilie L.
Pedersen, Per A.
Ellgaard, Lars
author_facet Virolainen, Minttu S.
Søltoft, Cecilie L.
Pedersen, Per A.
Ellgaard, Lars
author_sort Virolainen, Minttu S.
collection PubMed
description The human Fic domain-containing protein (FICD) is a type II endoplasmic reticulum (ER) membrane protein that is important for the maintenance of ER proteostasis. Structural and in vitro biochemical characterisation of FICD AMPylase and deAMPylase activity have been restricted to the soluble ER-luminal domain produced in Escherichia coli. Information about potentially important features, such as structural motifs, modulator binding sites or other regulatory elements, is therefore missing for the approximately 100 N-terminal residues including the transmembrane region of FICD. Expressing and purifying the required quantity and quality of membrane proteins is demanding because of the low yields and poor stability often observed. Here, we produce full-length FICD by combining a Saccharomyces cerevisiae-based platform with green fluorescent protein (GFP) tagging to optimise the conditions for expression, solubilisation and purification. We subsequently employ these conditions to purify milligram quantities of His-tagged FICD per litre of culture, and show that the purified, detergent-solubilised membrane protein is an active deAMPylating enzyme. Our work provides a straightforward methodology for producing not only full-length FICD, but also other membrane proteins in S. cerevisiae for structural and biochemical characterisation.
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spelling pubmed-89104942022-03-11 Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD Virolainen, Minttu S. Søltoft, Cecilie L. Pedersen, Per A. Ellgaard, Lars Int J Mol Sci Article The human Fic domain-containing protein (FICD) is a type II endoplasmic reticulum (ER) membrane protein that is important for the maintenance of ER proteostasis. Structural and in vitro biochemical characterisation of FICD AMPylase and deAMPylase activity have been restricted to the soluble ER-luminal domain produced in Escherichia coli. Information about potentially important features, such as structural motifs, modulator binding sites or other regulatory elements, is therefore missing for the approximately 100 N-terminal residues including the transmembrane region of FICD. Expressing and purifying the required quantity and quality of membrane proteins is demanding because of the low yields and poor stability often observed. Here, we produce full-length FICD by combining a Saccharomyces cerevisiae-based platform with green fluorescent protein (GFP) tagging to optimise the conditions for expression, solubilisation and purification. We subsequently employ these conditions to purify milligram quantities of His-tagged FICD per litre of culture, and show that the purified, detergent-solubilised membrane protein is an active deAMPylating enzyme. Our work provides a straightforward methodology for producing not only full-length FICD, but also other membrane proteins in S. cerevisiae for structural and biochemical characterisation. MDPI 2022-02-23 /pmc/articles/PMC8910494/ /pubmed/35269596 http://dx.doi.org/10.3390/ijms23052458 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Virolainen, Minttu S.
Søltoft, Cecilie L.
Pedersen, Per A.
Ellgaard, Lars
Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD
title Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD
title_full Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD
title_fullStr Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD
title_full_unstemmed Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD
title_short Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD
title_sort production of an active, human membrane protein in saccharomyces cerevisiae: full-length ficd
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8910494/
https://www.ncbi.nlm.nih.gov/pubmed/35269596
http://dx.doi.org/10.3390/ijms23052458
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