RESEARCH Over-Expression in E. coli and Purification of the Human OCTN2 Transport Protein Michele Galluccio • Linda Amelio • Mariafrancesca Scalise • Lorena Pochini • Eckhard Boles • Cesare Indiveri Published online: 13 April 2011 Ó Springer Science+Business Media, LLC 2011 Abstract The OCTN2 cDNA amplified from human skin fibroblast was cloned in pET-41a(?) carrying the gluta- thione S-transferase (GST) gene. The construct pET- 41a(?)–hOCTN2 was used to express the GST–hOCTN2 fusion protein in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained after 6 h of induction with IPTG at 28°C. The GST–hOCTN2 polypeptide was collected in the inclusion bodies and showed an apparent molecular mass on SDS-PAGE of 85 kDa. After solubili- zation with a buffer containing 0.8% sarkosyl and 3 M urea, the fusion protein was applied onto a Ni 2? -chelating chromatography column. The purified GST–hOCTN2 was treated with thrombin, and the hOCTN2 was separated from the GST by size exclusion chromatography. After the whole procedure, a yield of about 0.2 mg purified protein per liter of cell culture was obtained. To improve the protein yield, hOCTN2 cDNA was subjected to codon bias. The second codon CGG was substituted with AAA; the substitution led to the mutation R2K in the hOCTN2 pro- tein. hOCTN2(R2K) cDNA was cloned in pET-21a(?) carrying a C-terminal 6His tag. The resulting protein was expressed in E. coli Rosetta(DE3)pLysS and purified by Ni 2? -chelating chromatography. A yield of about 3.5 mg purified protein per liter of cell culture was obtained with this procedure. Keywords OCTN2 Á E. coli Á Over-expression Á Purification Á Codon bias Introduction Membrane proteins represent a significant fraction of pro- teomes of all organisms [1]. Among the membrane pro- teins, transporters play essential roles in living organisms since they are involved in nutrient uptake, elimination of catabolites, and regulation of the homeostasis of several cofactors and metabolites. That transport systems are essential for life is demonstrated by the occurrence of severe pathologies, which are caused by defects of trans- porter coding genes [2–7]. The chemico-physical proper- ties of membrane proteins, like insolubility in water and propensity to aggregate, make them difficult to handle. These properties limit both functional and structural studies of transporters and, in general, of membrane proteins. One of the main challenges of the study of membrane proteins from mammals is the difficulty in heterologous expression. This problem is more evident in bacterial hosts which, indeed, are the most suitable to obtain large scale prepa- rations of proteins for structural studies [8]. Recently, the M. Galluccio Á L. Amelio Á M. Scalise Á L. Pochini Á C. Indiveri (&) Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende, Italy e-mail: indiveri@unical.it M. Galluccio e-mail: mgalluccio@unical.it L. Amelio e-mail: linda.amelio@unical.it M. Scalise e-mail: mariafrancesca.scalise@unical.it L. Pochini e-mail: pochini@unical.it E. Boles Institute of Molecular Biosciences, Goethe-University Frankfurt am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany e-mail: e.boles@bio.uni-frankfurt.de 123 Mol Biotechnol (2012) 50:1–7 DOI 10.1007/s12033-011-9406-6