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Chem Phys Lipids. 2018 May;212:80-87. doi: 10.1016/j.chemphyslip.2018.01.006. Epub 2018 Jan 31.

Perdeuteration of cholesterol for neutron scattering applications using recombinant Pichia pastoris.

Author information

1
Institut Laue-Langevin, 71, Avenue des Martyrs, Grenoble 38042, France; Faculty of Natural Sciences, Keele University, Keele, Staffordshire ST5 5BG, United Kingdom.
2
acib, Austrian Centre of Industrial Biotechnology GmbH, 8010 Graz, Austria; Institute of Organic Chemistry, NAWI Graz, Graz University of Technology, 8010 Graz, Austria.
3
Institute of Molecular Biotechnology, NAWI Graz, BioTechMed Graz, Graz University of Technology, 8010 Graz, Austria.
4
Department of Biological Sciences and Institute of Structural and Molecular Biology, Birkbeck College, University of London, Malet Street, London WC1E 7HX, United Kingdom.
5
Centre for Neutron Scattering, Uppsala University, 751 20 Uppsala, Sweden.
6
Institut Laue-Langevin, 71, Avenue des Martyrs, Grenoble 38042, France.
7
acib, Austrian Centre of Industrial Biotechnology GmbH, 8010 Graz, Austria; Institute of Molecular Biotechnology, NAWI Graz, BioTechMed Graz, Graz University of Technology, 8010 Graz, Austria.
8
Biofilms - Research Centre for Biointerfaces and Biomedical Science Department, Faculty of Health and Society, Malmö University, Malmö 20506, Sweden.
9
Institut Laue-Langevin, 71, Avenue des Martyrs, Grenoble 38042, France. Electronic address: haertlein@ill.fr.

Abstract

Deuteration of biomolecules has a major impact on both quality and scope of neutron scattering experiments. Cholesterol is a major component of mammalian cells, where it plays a critical role in membrane permeability, rigidity and dynamics, and contributes to specific membrane structures such as lipid rafts. Cholesterol is the main cargo in low and high-density lipoprotein complexes (i.e. LDL, HDL) and is directly implicated in several pathogenic conditions such as coronary artery disease which leads to 17 million deaths annually. Neutron scattering studies on membranes or lipid-protein complexes exploiting contrast variation have been limited by the lack of availability of fully deuterated biomolecules and especially perdeuterated cholesterol. The availability of perdeuterated cholesterol provides a unique way of probing the structural and dynamical properties of the lipoprotein complexes that underly many of these disease conditions. Here we describe a procedure for in vivo production of perdeuterated recombinant cholesterol in lipid-engineered Pichia pastoris using flask and fed-batch fermenter cultures in deuterated minimal medium. Perdeuteration of the purified cholesterol was verified by mass spectrometry and its use in a neutron scattering study was demonstrated by neutron reflectometry measurements using the FIGARO instrument at the ILL.

KEYWORDS:

Cholesterol; Lipid engineering; Neutron scattering; Perdeuteration; Pichia pastoris

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