Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state.

Autor: Aduri NG; Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark., Ernst HA; Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark., Prabhala BK; Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark., Bhatt S; Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark., Boesen T; Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus, Denmark., Gajhede M; Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark., Mirza O; Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address: om@sund.ku.dk.
Jazyk: angličtina
Zdroj: Biochemical and biophysical research communications [Biochem Biophys Res Commun] 2018 Jan 08; Vol. 495 (2), pp. 1738-1743. Date of Electronic Publication: 2017 Dec 05.
DOI: 10.1016/j.bbrc.2017.12.008
Abstrakt: The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.
(Copyright © 2017 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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