Submitted on August 14, 2006
Revised on October 18, 2006
Accepted on October 27, 2006
Ratio determination of plasma wild-type and mutant apoA-I using mass spectrometry: quantification, purification and expression of L159R apoA-I (apoA-IFin)
John S. Owen, Manish S. Bharadwaj, Michael J. Thomas, Shaila Bhat, Michael P. Samuel, and Mary G. Sorci-Thomas
Dept of Pathology, Wake Forest University Medical Center, Winston-Salem, NC 27157
Corresponding Author: msthomas{at}wfubmc.edu
In this report, methods are described to isolate milligram quantities of a mutant 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. Employing an E.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-IFin) from inclusion bodies. In addition, we describe a mass spectrometric method for measuring the L159R to wild-type apoA-I ratio in a one-microliter plasma sample. These new methods will facilitate further studies into the mechanism explaining the dominant negative phenotype associated with the expression of the L159R apoA-I protein in humans.
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