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Streamlined purification of fluorescently labeled Escherichia coli phosphate-binding protein (PhoS) suitable for rapid-kinetics applications.
Smith, Dustin D; Girodat, Dylan; Wieden, Hans-Joachim; Selinger, L Brent.
Afiliação
  • Smith DD; University of Lethbridge, Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, 4401 University Dr. W, Lethbridge, AB, T1K 3M4, Canada; University of Lethbridge, Department of Biological Sciences, 4401 University Dr. W, Lethbridge, AB, T1K 3M4, Canada. Electronic add
  • Girodat D; University of Lethbridge, Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, 4401 University Dr. W, Lethbridge, AB, T1K 3M4, Canada. Electronic address: dylan.girodat@uleth.ca.
  • Wieden HJ; University of Lethbridge, Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, 4401 University Dr. W, Lethbridge, AB, T1K 3M4, Canada. Electronic address: hj.wieden@uleth.ca.
  • Selinger LB; University of Lethbridge, Department of Biological Sciences, 4401 University Dr. W, Lethbridge, AB, T1K 3M4, Canada. Electronic address: selibl@uleth.ca.
Anal Biochem ; 537: 106-113, 2017 11 15.
Article em En | MEDLINE | ID: mdl-28941789
ABSTRACT
Fluorescently labeled phosphate-binding proteins can be used as biomolecular tools to measure the release of inorganic phosphate (Pi) from enzymes in real time, enabling the detailed kinetic analysis of dephosphorylating enzymes using rapid-kinetics approaches. Previously reported methods to purify fluorescently labeled phosphate-binding proteins (PhoS) from Escherichia coli are laborious, and a simplified approach is needed. Here, we report the characterization of a cytosol-localized variant (A197C) of PhoS that allows a streamlined purification for subsequent covalent conjugation with a fluorescent dye. We show that export of PhoS into the periplasmic space is not required for the fluorescence-based detection of Pi binding. Furthermore, we report the addition of a C-terminal His-tag, simplifying the purification of PhoS from the cytosol via Ni2+-affinity chromatography, yielding a fully functional fusion protein (HC PhoS A197C). We demonstrate the utility of fluorescently labeled HC PhoS A197C for rapid-kinetics applications by measuring, using stopped-flow, the Pi release kinetics from LepA/EF4 following 70S ribosome-stimulated GTP hydrolysis. Altogether, the approach developed here allows for the high-yield and simplified in-house production of a Pi detection system suitable for rapid-kinetics approaches with comparable sensitivity to the commercially available Phosphate Sensor.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Espectrometria de Fluorescência / Técnicas de Química Analítica / Proteínas de Escherichia coli / Proteínas de Ligação a Fosfato / Escherichia coli / Corantes Fluorescentes Idioma: En Ano de publicação: 2017 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Espectrometria de Fluorescência / Técnicas de Química Analítica / Proteínas de Escherichia coli / Proteínas de Ligação a Fosfato / Escherichia coli / Corantes Fluorescentes Idioma: En Ano de publicação: 2017 Tipo de documento: Article