1. 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.
- Author
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Horn JVC, Ellena RA, Tran JJ, Beck WHJ, Narayanaswami V, and Weers PMM
- 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
- 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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