HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, H.-h. Articles by Sorci-Thomas, M. G. Search for Related Content PubMed PubMed Citation Articles by Li, H.-h. Articles by Sorci-Thomas, M. G. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 42, 2084-2091, December 2001
Copyright © 2001 by Lipid Research, Inc.

Methods

Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL

Correspondence to: Mary G. Sorci-Thomas, at the Department of Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157., msthomas{at}wfubmc.edu (E-mail)

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., M. J. Thomas, W. Pan, E. Alexander, M. Samuel, and M. G. Sorci-Thomas. Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL. J. Lipid Res. 2001. 42: 2084–2091.

Supplementary key words: recombinant apoA-I, protein expression of mature apoA-I, fluorescent probes, Eschericha coli expression, intein, LCAT activity, recombinant discoidal HDL, rhodamine and fluorescein probe attachment

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Z. Wu, V. Gogonea, X. Lee, M. A. Wagner, X.-M. Li, Y. Huang, A. Undurti, R. P. May, M. Haertlein, M. Moulin, et al. Double Superhelix Model of High Density Lipoprotein J. Biol. Chem., December 25, 2009; 284(52): 36605 - 36619. [Abstract] [Full Text] [PDF]

				M. J. Thomas, S. Bhat, and M. G. Sorci-Thomas Three-dimensional models of HDL apoA-I: implications for its assembly and function J. Lipid Res., September 1, 2008; 49(9): 1875 - 1883. [Abstract] [Full Text] [PDF]

				J. S. Owen, M. S. Bharadwaj, M. J. Thomas, S. Bhat, M. P. Samuel, and M. G. Sorci-Thomas Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin J. Lipid Res., January 1, 2007; 48(1): 226 - 234. [Abstract] [Full Text] [PDF]

				B Y Ishida, K G Duncan, K R Bailey, J P Kane, and D M Schwartz High density lipoprotein mediated lipid efflux from retinal pigment epithelial cells in culture Br J Ophthalmol, May 1, 2006; 90(5): 616 - 620. [Abstract] [Full Text] [PDF]

				S. Bhat, M. G. Sorci-Thomas, E. T. Alexander, M. P. Samuel, and M. J. Thomas Intermolecular Contact between Globular N-terminal Fold and C-terminal Domain of ApoA-I Stabilizes Its Lipid-bound Conformation: STUDIES EMPLOYING CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY J. Biol. Chem., September 23, 2005; 280(38): 33015 - 33025. [Abstract] [Full Text] [PDF]

				S. Bhat, M. Zabalawi, M. C. Willingham, G. S. Shelness, M. J. Thomas, and M. G. Sorci-Thomas Quality control in the apoA-I secretory pathway: deletion of apoA-I helix 6 leads to the formation of cytosolic phospholipid inclusions J. Lipid Res., July 1, 2004; 45(7): 1207 - 1220. [Abstract] [Full Text] [PDF]

				H.-h. Li, D. S. Lyles, W. Pan, E. Alexander, M. J. Thomas, and M. G. Sorci-Thomas ApoA-I Structure on Discs and Spheres. VARIABLE HELIX REGISTRY AND CONFORMATIONAL STATES J. Biol. Chem., October 11, 2002; 277(42): 39093 - 39101. [Abstract] [Full Text] [PDF]