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Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD.
Virolainen, Minttu S; Søltoft, Cecilie L; Pedersen, Per A; Ellgaard, Lars.
Afiliación
  • Virolainen MS; Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark.
  • Søltoft CL; Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark.
  • Pedersen PA; Department of Biology, University of Copenhagen, Universitetsparken 15, DK-2200 Copenhagen, Denmark.
  • Ellgaard L; Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark.
Int J Mol Sci ; 23(5)2022 Feb 23.
Article en En | MEDLINE | ID: mdl-35269596
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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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Saccharomyces cerevisiae / Proteínas de Saccharomyces cerevisiae Límite: Humans Idioma: En Revista: Int J Mol Sci Año: 2022 Tipo del documento: Article País de afiliación: Dinamarca Pais de publicación: Suiza

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Saccharomyces cerevisiae / Proteínas de Saccharomyces cerevisiae Límite: Humans Idioma: En Revista: Int J Mol Sci Año: 2022 Tipo del documento: Article País de afiliación: Dinamarca Pais de publicación: Suiza