John Owen - Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry:  tools for studying apoA-IFin.

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  Publication Details (including relevant citation   information):

  J Lipid Res 48:226-234, 2007


  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.

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