Faculty Collaboration Database

Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin. J Lipid Res 2007 Jan;48(1):226-34

Date

10/31/2006

Pubmed ID

17071967

DOI

10.1194/jlr.D600031-JLR200

Scopus ID

2-s2.0-33845972961 (requires institutional sign-in at Scopus site)   10 Citations

Abstract

In this report, methods are described to isolate milligram quantities of a mutant apolipoprotein A-I (apoA-I) protein for use in structure-function studies. Expression of the L159R apoA-I mutation in humans reduces the concentration of plasma wild-type apoA-I, thus displaying a dominant negative phenotype in vivo. Earlier attempts to express and isolate this mutant protein resulted in extensive degradation and protein misfolding. Using an Escherichia coli expression system used predominantly for the isolation of soluble apoA-I mutant proteins, we describe the expression and purification of L159R apoA-I (apoA-I(Fin)) from inclusion bodies. In addition, we describe a mass spectrometric method for measuring the L159R-to-wild-type apoA-I ratio in a 1 microl plasma sample. These new methods will facilitate further studies into the mechanism behind the dominant negative phenotype associated with the expression of the L159R apoA-I protein in humans.

Author List

Owen JS, Bharadwaj MS, Thomas MJ, Bhat S, Samuel MP, 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

Amino Acid Substitution
Apolipoprotein A-I
Escherichia coli
Humans
Mass Spectrometry
Phenotype
Plasmids
Recombinant Proteins