Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3 Mariafrancesca Scalise 1 , Michele Galluccio 1 , Lorena Pochini, Cesare Indiveri ⇑ Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende, Italy article info Article history: Received 19 April 2012 Available online xxxx Keywords: OCTN3 Expression Purification Transport Reconstitution Liposomes 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 insol- uble fraction of the cell lysate and purified by Ni 2+ -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 lipo- somes. The protein catalyzed a time dependent [ 3 H]carnitine uptake which was stimulated by intralipos- omal ATP and nearly independent of the pH. The K m for carnitine was 36 lM. [ 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. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Membrane transport systems allow uptake and efflux of a very large variety of molecules such as nutrients, catabolites and cofac- tors or their precursors [1–5]. Genes coding membrane transport- ers, indeed, represent a significant fraction of the genome of all organisms [6]. The occurrence of severe pathologies caused by ge- netic defects demonstrates the central role of these proteins in maintaining cell homeostasis [5,7]. In spite of their relevance, func- tional and structural studies on higher animal membrane trans- porters only recently have largely spread out. The study of these proteins, is challenged by the difficulties in expressing them in bacterial hosts and, hence, in purifying and studying functional properties in in vitro experimental systems [8]. Recently, over- expression in Escherichia coli of the human OCTN1 and OCTN2 transporters [9,10] was obtained. The hOCTN1 recombinant protein was then reconstituted in liposomes and functionally char- acterized [11,12]. The two transporters belong to a small protein sub-family, OCTN (Organic Cation Transporters Novel), to which a third member, OCTN3 also belongs. The coding genes for OCTN’s have been identified only in higher mammals. Differently from OCTN1 and 2, the gene of OCTN3 has not yet been annotated in Homo sapiens genome [13,14], even though, the OCTN3 protein has been detected in human skin and sperm using antibody against mouse (m)OCTN3 [15–18]. The functional information concerning the OCTN3 transporter comes from experiments performed with murine cells [16,19–21] or in cell lines in which the transporter was artificially expressed [13]. The data obtained in the cell sys- tems show that the OCTN3 is a carnitine transporter [19–21] and that it also recognizes acetylcarnitine [16]. In the present work the over-expression of mOCTN3 and its reconstitution in liposomes was obtained. 2. Materials and methods 2.1. Materials E. coli Rosetta(DE3) and pET-21a(+) were purchased from Nova- gen; full length cDNA (IRCLp5011H1113D) from Source BioScience UK Limited; cloning reagents from Fermentas; yeast extract from Applichem; Triptone, Amberlite XAD-4, 3-sn-phosphatidylcoline from egg yolk, Triton-X100, Sephadex G-75, His-select Ni-affinity gel, protease inhibitor cocktail and L-carnitine from Sigma; L-[N- methyl-3H]carnitine hydrochloride from PerkinElmer. All the other reagents were of analytical grade. 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.04.105 Abbreviations: TEA, tetraethylammonium; PLP, pyridoxal 5-phosphate; MTSET, [2- (trimethylammonium)ethyl]methanethiosulfonate; p-OHMB, p-hydroxymercuriben- zoate; GABA, c-aminobutyric acid; ANTP, adenosine 5 0 -(b,c-imido)triphosphate. ⇑ Corresponding author. Address: Dipartimento di Biologia Cellulare, Università della Calabria, Via P. Bucci cubo 4c, 87036 Arcavacata di Rende, CS, Italy. Fax: +39 0984 492911. E-mail address: indiveri@unical.it (C. Indiveri). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Please cite this article in press as: M. Scalise et al., Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3, Biochem. Biophys. Res. Commun. (2012), http://dx.doi.org/10.1016/j.bbrc.2012.04.105