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.

Highlights

  • Apolipoprotein A-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

  • We demonstrate the use of this system for the rapid generation of cysteine-containing mutant Apolipoprotein A-I (apoA-I) proteins, which are labeled with fluorescent probes and used to make recombinant HDL (rHDL) for fluorescence resonance energy transfer (FRET) studies

  • isopropyl ␤-thiogalactopyranoside (IPTG) induction of the apoA-I-inteinchitin binding domain fusion protein (AI-CBD) (Fig. 1A, lane 3) shows a predominant band at 83 kDa in the crude bacterial cell extract that was not present in cells grown in the absence of IPTG (Fig. 1A, lane 2)

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Summary

Introduction

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. 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. Cysteinecontaining 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. Discoidal rHDL were 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. Preparation and incorporation of probelabeled apoA-I for fluorescence resonance energy transfer studies of rHDL.

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