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. 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. 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.

Apolipoprotein A-I Helix 6 Negatively Charged Residues Attenuate Lecithin−Cholesterol Acyltransferase (LCAT) Reactivity

“…After the final purification, lyophilized mutant apoA-I was taken up in 6 M guanidine hydrochloride and dialyzed against 10 mM ammonium bicarbonate, pH 7.4, containing 3 μM EDTA and 15 μM sodium azide. The protein purity and molecular weight were determined using a Quattro II mass spectrometer as previously reported ( , ).…”

High-Density Lipoprotein Biogenesis: Defining the Domains Involved in Human Apolipoprotein A-I Lipidation

“…Two cysteines will form a disulfide bond, a process called locking, if they are 3–5 Å apart and no steric hindrance is present to impede bond formation. To study lipidation and the role of helix opening, two cysteines were added to apoA-I using standard techniques − to create mutant proteins that could undergo intramolecular locking, which prevents regions from moving apart. Several double-cysteine-containing mutants were prepared that, based on our model, would not form intramolecular disulfide bonds.…”

“…The coding sequence for wild-type and mutant apoA-I was cloned from the CMV5 vector by Custom DNA Constructs and amplified by PCR as described and inserted into the pTYB11 vector. Expression and purification of the double-cysteine-containing apoA-I mutants from inclusion bodies were conducted as previously described. − Mutant apoA-Is released from a chitin affinity column with dithiothreitol (DTT) are monomeric and essentially pure as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Protein purities and molecular weights were determined by mass spectrometry of the intact proteins and are listed in Table .…”

“…The last traces of solvent were removed under vacuum for 18 h. POPC was added to a solution containing sodium deoxycholate at a deoxycholate:POPC molar ratio of 2:1 by incubation at 37 °C with intermittent shaking for 1 h and then vigorously shaken on a standard multitube vortexer (VWR Scientific) for 1 h at room temperature. The crude rHDL was purified by application to two Superdex 200 HR 10/30 columns linked in tandem and eluted at 0.5 mL/min as previously described. , The purified rHDL was then analyzed on 4 to 30% nondenaturing gels (CBS Scientific) to assess yield and particle size. , …”

“…The crude rHDL was purified by application to two Superdex 200 HR 10/30 columns linked in tandem and eluted at 0.5 mL/min as previously described. 61,64 The purified rHDL was then analyzed on 4 to 30% nondenaturing gels (CBS Scientific) to assess yield and particle size. 34,36 Cell Culture and Purification of Nascent HDL.…”