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 (Myr2GroPCho) 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 alpha-helical content of both plasma and recombinant apoA-II increased similarly when the proteins associated with Myr2GroPCho vesicles, at the expense of a random-coil structure. Lipid-bound apoA-II consisted of 70-72% alpha helices, suggesting the presence of three 18-residue alpha helices/apoA-II monomer. Cross-linking experiments indicated that Myr2GroPCho 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 Myr2GroPCho 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.
Lopez
J
J
URA CNRS 1283, Institut des Cordeliers, Paris, France.
Latta
M
M
Collet
X
X
Vanloo
B
B
Jung
G
G
Denefle
P
P
Rosseneu
M
M
Chambaz
J
J
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism; D016633:Apolipoprotein A-II / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001667:Binding, Competitive; D004134:Dimyristoylphosphatidylcholine; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008075:Lipoproteins, HDL / Q000378:metabolism; D008081:Liposomes; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012549:Schistosoma japonicum / Q000201:enzymology / Q000235:genetics
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D008075:Lipoproteins, HDL; D008081:Liposomes; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D004134:Dimyristoylphosphatidylcholine