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

Author

Mary Sorci Thomas PhD Professor in the Medicine 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