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A protocol for the combined sub-fractionation and delipidation of lipid-binding proteins by hydrophobic interaction chromatography

Velkov, Tony, Lim, Maria L. R., Capuanoa, Benjamin and Prankerd, Richard 2008, A protocol for the combined sub-fractionation and delipidation of lipid-binding proteins by hydrophobic interaction chromatography, Journal of chromotography B, vol. 867, no. 2, pp. 238-246, doi: 10.1016/j.jchromb.2008.04.011.

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Title A protocol for the combined sub-fractionation and delipidation of lipid-binding proteins by hydrophobic interaction chromatography
Author(s) Velkov, Tony
Lim, Maria L. R.
Capuanoa, Benjamin
Prankerd, Richard
Journal name Journal of chromotography B
Volume number 867
Issue number 2
Start page 238
End page 246
Publisher Elsevier
Place of publication Amsterdam, Netherlands
Publication date 2008-05-15
ISSN 1570-0232
1873-376X
Keyword(s) delipidation
hydrophobic interaction chromatography;
lipidex
fatty acid binding protein
peroxisome proliferator activated receptor;
Summary Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor gamma ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.
Language eng
DOI 10.1016/j.jchromb.2008.04.011
Field of Research 030499 Medicinal and Biomolecular Chemistry not elsewhere classified
HERDC Research category C1.1 Refereed article in a scholarly journal
ERA Research output type C Journal article
HERDC collection year 2008
Copyright notice ©2008, Elsevier B.V.
Persistent URL http://hdl.handle.net/10536/DRO/DU:30018870

Document type: Journal Article
Collection: School of Medicine
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Created: Fri, 11 Sep 2009, 10:14:23 EST by Rachael Wilson

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