title: Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: the mouse carnitine transporter OCTN3.
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.
authors.affiliation: Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende, Italy.
authors.forename: Mariafrancesca
authors.initials: M
authors.lastname: Scalise
authors.forename: Michele
authors.initials: M
authors.lastname: Galluccio
authors.forename: Lorena
authors.initials: L
authors.lastname: Pochini
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
mesh_terms: D000818:Animals; D002331:Carnitine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry; D051379:Mice; D027701:Organic Cation Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D008081:Liposomes; D027701:Organic Cation Transport Proteins; D011994:Recombinant Proteins; C498687:Slc22a21 protein, mouse; D002331:Carnitine