Purification and Characterization of Recombinant Human Apolipoprotein A‐II Expressed in Escherichia coli

We have expressed recombinant human apolipoprotein A‐II (apoA‐II) in Escherichia coli , as a fusion protein with Schistosoma japonicum glutathione‐ S ‐transferase (GST). The GST – AII fusion protein was recovered by affinity chromatography using glutathione as a ligand. After thrombin cleavage and removal of the GST carrier, recombinant apoA‐II was obtained in a highly purified form and was exclusively composed of dimeric apoA‐II. Kinetics of association to dimyristoylglycerophosphocholine (Myr 2 GroPCho) vesicles showed that recombinant apoA‐II exhibited the same pattern of association as human plasma apoA‐II. Electron microscopic analysis of the complexes showed a typical pattern of rouleaux, characteristic of stacked discs, with a diameter similar to that determined by gradient‐gel electrophoresis. Circular dichroism measurements showed that the α‐helical content of both plasma and recombinant apoA‐II increased similarly when the proteins associated with Myr 2 GroPCho vesicles, at the expense of a random‐coil structure. Lipid‐bound apoA‐II consisted of 70–72% α helices, suggesting the presence of three 18‐residue α helices/apoA‐II monomer. Cross‐linking experiments indicated that Myr 2 GroPCho complexes contained two molecules dimeric apoA‐II/vesicle. Recombinant apoA‐II was as efficient as plasma apoA‐II in associating with HDL subclasses, and in displacing apoA‐I from dipalmitoylglycerophosphocholine/cholesterol/apoA‐I complexes, most likely due to its highly ordered secondary structure when associated with Myr 2 GroPCho vesicles. These findings demonstrate that recombinant apoA‐II exhibits the same structural and functional properties as human plasma apoA‐II. Thus, the expression system utilized is appropriate to produce mutagenized forms to further structure/function analysis.