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

Abstract

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.

Keywords

• recombinant apoA-I
• protein expression of mature apoA-I
• fluorescent probes
• Eschericha coli expression
• intein
• LCAT activity