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Characterization of the C‐Terminal Domain of Human Apolipoprotein A‐I via a Novel Apolipoprotein Chimera
Author(s) -
Horn James,
Ellena Rachel,
Tran Jesse,
Narayanaswami Vasanthy,
Weers Paul
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.886.10
Subject(s) - chimera (genetics) , apolipoprotein b , circular dichroism , chemistry , biochemistry , biophysics , recombinant dna , biology , cholesterol , gene
Human apolipoprotein A‐I (apoA‐I) is a 28 kDa exchangeable apolipoprotein and is the major protein component of high‐density lipoproteins, playing a critical role in reverse cholesterol transport. ApoA‐I has a two‐domain structure: an α‐helical N‐terminal domain (NT, residues 1‐189) and a less structured C‐terminal domain (CT, residues 190‐243). To examine the properties of the CT domain of apoA‐I independent from the NT domain, a novel chimeric apolipoprotein was engineered by attaching residues 179 to 243 of apoA‐I to apolipophorin III (apoLp‐III). This insect apolipoprotein is used as a model and has a one domain structure made of a bundle of α‐helices . The apoLp‐III/apoA‐I chimera was expressed in E. coli BL21 DE3 cells and purified using nickel affinity chromatography. Western blot analysis confirmed the presence of apoA‐I within the chimera. ApoLp‐III is monomeric but cross‐linking studies with dimethyl suberimidate showed that the chimera self‐associates similar to apoA‐I. Far UV circular dichroism showed that the chimera displayed less α‐helical content compared to apoLp‐III which is consistent with the addition of an unstructured domain. Denaturation analysis using guanidine HCl showed that the protein stability of the chimera was increased compared to apoLp‐III. Functional analysis showed that the chimera was able to solubilize phospholipid vesicles at a much faster rate compared to apoLp‐III. Since the addition of the CT domain of apoA‐I to apoLp‐III resulted in oligomerization and enhanced lipid binding, these properties reside in the CT domain of apoA‐I and could be transferred to apoLp‐III. This demonstrates the potential to use the chimera as a tool to study the CT domain of apoA‐I.