Faculty Collaboration Database

Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL. J Lipid Res 2001 Dec;42(12):2084-91

Date

12/06/2001

Pubmed ID

11734582

Scopus ID

2-s2.0-0035664834 (requires institutional sign-in at Scopus site)   19 Citations

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.

Author List

Li HH, Thomas MJ, Pan W, Alexander E, Samuel M, Sorci-Thomas MG

Authors

Mary Sorci Thomas PhD Professor in the Medicine department at Medical College of Wisconsin
Michael J. Thomas PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Apolipoprotein A-I
Binding Sites
Chitin
Cysteine
Energy Transfer
Escherichia coli
Fluorescent Dyes
Lipoproteins, HDL
Mutagenesis, Site-Directed
Mutation
Particle Size
Phosphatidylcholine-Sterol O-Acyltransferase
Protein Structure, Tertiary
Recombinant Fusion Proteins
Spectrometry, Fluorescence
Spectrometry, Mass, Electrospray Ionization