title: A simple approach for human recombinant apolipoprotein E4 expression and purification.
abstract: We report a simple expression and purification procedure for the production of recombinant apolipoprotein E4 (apoE4), an important protein for the lipid homeostasis in humans that plays critical roles in the pathogenesis of cardiovascular and neurodegenerative diseases. Our approach is based on the expression of a thioredoxin-apoE4 fusion construct in bacterial cells and subsequent removal of the fused thioredoxin using the highly specific 3C protease, avoiding costly and laborious lipidation-delipidation steps used before. Our approach results in rapid, high-yield production of structurally and functionally competent apoE4 as evidenced by secondary structure measurements, thermal and chemical melting profiles and the kinetic profile of solubilization of dimyristoyl-phosphatidylcholine (DMPC) vesicles. This protocol is appropriate for laboratories with little experience in apolipoprotein biochemistry and will facilitate future studies on the role of apoE4 in the pathogenesis of cardiovascular disease and neurodegenerative diseases, including Alzheimer's disease.
authors.affiliation: Institute of Biology, National Center for Scientific Research Demokritos, Agia Paraskevi, Athens 15310, Greece.
authors.forename: Letta
authors.initials: L
authors.lastname: Argyri
authors.forename: Vassiliki
authors.initials: V
authors.lastname: Skamnaki
authors.forename: Efstratios
authors.initials: E
authors.lastname: Stratikos
authors.forename: Angeliki
authors.initials: A
authors.lastname: Chroni
mesh_terms: D000256:Adenoviridae; D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D053327:Apolipoprotein E4 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001254:Astrocytoma / Q000235:genetics / Q000378:metabolism; D002318:Cardiovascular Diseases / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D010713:Phosphatidylcholines / Q000378:metabolism; D010957:Plasmids; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D014169:Transformation, Bacterial
chemical_list: D053327:Apolipoprotein E4; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins