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

By James V.C. Horn, Rachel A. Ellena, Jesse J. Tran, Wendy H.J. Beck, Vasanthy Narayanaswami, Paul M.M. Weers

July 28, 2017

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Title

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

James V.C. Horn, Rachel A. Ellena, Jesse J. Tran, Wendy H.J. Beck, Vasanthy Narayanaswami, Paul M.M. Weers

Year

2017

Journal

Biochimica et Biophysica Acta (BBA) - Biomembranes

Abstract

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18 kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28 kDa 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.

Full Article

http://www.sciencedirect.com/science/article/pii/S0005273617301335

Instrument

J-810

Keywords

Circular dichroism, Secondary structure, Chemical stability, Protein denaturation, Biochemistry