title: Biochemical and biophysical characterization of recombinant rat apolipoprotein E: similarities to human apolipoprotein E3.
abstract: Apolipoprotein E (apoE) is an anti-atherogenic protein that plays a critical role in maintaining plasma cholesterol and triglyceride homeostasis by virtue of its ability to act as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. In this study, we characterized the biochemical and biophysical features of recombinant rat apoE, in comparison with those of human apoE3. Rat apoE was overexpressed in Escherichia coli using a codon optimized system and purified by affinity chromatography. SDS-PAGE and RP-HPLC of rat apoE confirmed the purity, while immunoblot verified the identity and cross-reactivity with the LDLr-binding region of apoE3. The α-helical content was calculated to be ~45% by circular dichroism spectroscopy. The protein exists in a predominantly tetrameric form in lipid-free state. Chemical denaturation studies reveal that the unfolding pattern is biphasic with mid points of denaturation corresponding to 0.8 and 2.2 M guanidine hydrochloride, suggesting the presence of two domains. Rat apoE converts DMPC vesicles to smaller DMPC/apoE complexes with a first order rate constant of 0.12 min(-1). It has the ability to bind the LDLr and to heparin. Our studies indicate that although its sequence resembles apoE4, an isoform of apoE3, rat apoE displays the biophysical behavior of apoE3.
authors.affiliation: Department of Chemistry and Biochemistry, 1250 Bellflower Boulevard, California State University Long Beach, Long Beach, CA 90840, USA.
authors.forename: Tuyen N
authors.initials: TN
authors.lastname: Tran
authors.forename: Sea H
authors.initials: SH
authors.lastname: Kim
authors.forename: Carlos
authors.initials: C
authors.lastname: Gallo
authors.forename: Max
authors.initials: M
authors.lastname: Amaya
authors.forename: Jessica
authors.initials: J
authors.lastname: Kyees
authors.forename: Vasanthy
authors.initials: V
authors.lastname: Narayanaswami
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053318:Apolipoprotein E3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053327:Apolipoprotein E4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D051381:Rats; D011973:Receptors, LDL / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein
chemical_list: D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D008024:Ligands; D011973:Receptors, LDL; D011994:Recombinant Proteins; D006493:Heparin; D004134:Dimyristoylphosphatidylcholine