Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: the mouse carnitine transporter OCTN3

Biochemical and Biophysical Research Communications
Mariafrancesca ScaliseCesare Indiveri

Abstract

pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni(2+)-chelating chromatography with a yield of 2mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [(3)H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K(m) for carnitine was 36 μM. [(3)H]carnitine transport was inhibited by carnitine analogues and some Cys and NH(2) reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.

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Citations

Jan 3, 2016·Biochimica Et Biophysica Acta·Lorena PochiniCesare Indiveri
Oct 24, 2015·FEBS Letters·Mariafrancesca ScaliseCesare Indiveri
Aug 11, 2015·The International Journal of Biochemistry & Cell Biology·Lara NapolitanoCesare Indiveri
Apr 12, 2015·Biochimica Et Biophysica Acta·Lara ConsoleCesare Indiveri
Dec 11, 2014·Toxicological Sciences : an Official Journal of the Society of Toxicology·Michele GalluccioCesare Indiveri
Mar 2, 2018·Scientific Reports·Lara ConsoleCesare Indiveri
Jun 19, 2013·Journal of Biomolecular Screening·Lorena PochiniCesare Indiveri
Feb 12, 2020·International Journal of Molecular Sciences·Lorena PochiniCesare Indiveri

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