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Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity.
- Source :
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Biochimica et biophysica acta. Biomembranes [Biochim Biophys Acta Biomembr] 2017 Aug; Vol. 1859 (8), pp. 1317-1325. Date of Electronic Publication: 2017 Apr 21. - Publication Year :
- 2017
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Abstract
- Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III.<br /> (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
Apolipoprotein A-I genetics
Apolipoproteins genetics
Binding Sites
Cloning, Molecular
Escherichia coli genetics
Escherichia coli metabolism
Gene Expression
Grasshoppers chemistry
Humans
Insect Proteins genetics
Kinetics
Lipid Droplets chemistry
Models, Molecular
Phosphatidylglycerols chemistry
Protein Binding
Protein Conformation, alpha-Helical
Protein Engineering
Protein Interaction Domains and Motifs
Protein Multimerization
Protein Stability
Recombinant Fusion Proteins genetics
Recombinant Proteins chemistry
Recombinant Proteins genetics
Solubility
Thermodynamics
Type C Phospholipases chemistry
Apolipoprotein A-I chemistry
Apolipoproteins chemistry
Insect Proteins chemistry
Lipopolysaccharides chemistry
Lipoproteins, LDL chemistry
Recombinant Fusion Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0005-2736
- Volume :
- 1859
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Biochimica et biophysica acta. Biomembranes
- Publication Type :
- Academic Journal
- Accession number :
- 28434970
- Full Text :
- https://doi.org/10.1016/j.bbamem.2017.04.017