Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL.
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
HH
Department of Pathology, The Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA.
Thomas
M J
MJ
Pan
W
W
Alexander
E
E
Samuel
M
M
Sorci-Thomas
M G
MG
D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004735:Energy Transfer; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010316:Particle Size; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D016632:Apolipoprotein A-I; D005456:Fluorescent Dyes; D008075:Lipoproteins, HDL; D011993:Recombinant Fusion Proteins; D002686:Chitin; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D003545:Cysteine