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 Ni2+-chelating chromatography with a yield of 2 mg/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 [H-3]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K-m for carnitine was 36 mu M. [H-3]carnitine transport was inhibited by carnitine analogues and some Cys and NH2 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. (C) 2012 Elsevier Inc. All rights reserved.

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

Scalise M;GALLUCCIO, Michele;POCHINI, Lorena;INDIVERI, Cesare
2012

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 Ni2+-chelating chromatography with a yield of 2 mg/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 [H-3]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K-m for carnitine was 36 mu M. [H-3]carnitine transport was inhibited by carnitine analogues and some Cys and NH2 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. (C) 2012 Elsevier Inc. All rights reserved.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11770/134698
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