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Journal of Lipid Research, Vol. 42, 2084-2091, December 2001
Copyright © 2001 by Lipid Research, Inc.

Methods

Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL

Correspondence to: Mary G. Sorci-Thomas, at the Department of Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157., msthomas{at}wfubmc.edu (E-mail)

Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester. Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of lipid-bound apoA-I. Herein, we report new methodologies allowing rapid expression, fluorescent labeling, and recombinant HDL (rHDL) preparation for use in apoA-I in fluorescence resonance energy transfer (FRET) studies. Cysteine-containing mutant forms of human apoA-I were cloned into the pTYB12 vector containing a T7 promoter, a modified self-splicing protein element (intein), and a small affinity tag [chitin binding domain (CBD)]. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates, and bound to a chitin-affinity column. Release of mature human apoA-I was initiated by the addition of DTT, which induced self-cleavage at the COOH terminus of the intein - CBD fusion protein. ApoA-I was further purified by Q-sepharose and then used for fluorescent probe labeling.

Discoidal rHDL were then prepared with donor and/or acceptor labeled apoA-I and characterized with respect to their size, composition and ability to activate LCAT. — Li, H-h., M. J. Thomas, W. Pan, E. Alexander, M. Samuel, and M. G. Sorci-Thomas. Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL. J. Lipid Res. 2001. 42: 2084–2091.

Supplementary key words: recombinant apoA-I, protein expression of mature apoA-I, fluorescent probes, Eschericha coli expression, intein, LCAT activity, recombinant discoidal HDL, rhodamine and fluorescein probe attachment

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