Espressione di sistemi di trasporto umani di membrana plasmatica in E.coli

Abstract

In this work we studied the bacterial over-expression of two human transporters belonging the Organi Cation Trasportr Novel (OCTN) protein family. This family includes the mammalian plasma membrane transporters OCTN1, OCTN2 and OCTN3, which share more than 66 % identity with each other. Defects of OCTN1 and OCTN2 are associated with the Crohn disease and the primary carnitine deficiency, respectively. The molecular and functional studies of these transport systems are hampered by the difficulties in bacterial overexpression and solubilization. Thus, the transporters have been functionally characterized in cell systems like oocytes expressing the protein at low level (Lahjouji et al 2001; Tamai et al 1997) or in proteoliposomes reconstituted with the protein extracted from kidney cells (Pochini et al 2004). On the one hand the OCTN2 has been well characterized. On the other hand many discrepancies about the function of OCTN1 have been reported (Lahjouji et al 2001; Tamai et al 1997; Yabuuchi et al 1999). OCTN2 catalyzes a sodium-dependent antiport of carnitine with itself or carnitine derivatives. It was found that some drugs, such as omeprazole, can interact with the transporter (Pochini et al 2009). However, so far, no examples of strategies of large scale over-expression and/or purification of these proteins have been reported and, hence, no structural data are available. Here, the first successful procedures for the large scale over-expression and purification of the transporters are described. The hOCTN1 cDNA, previous obtained from primary human fibroblasts, was cloned in different plasmids, and the best over-expression was obtained with the pH6EX3 in RosettaGami2(DE3)pLysS strain. The expressed protein results as a 6-His tagged protein. Also the hOCTN2 cDNA was cloned in several plasmids but the recombinant protein was obtained only using pET41 in E.coli 5 Rosetta(DE3)pLysS. By this strategy, the OCTN2 was expressed as GST-6His tagged protein. The over-expressed OCTN1 and OCTN2 had an apparent molecular mass of 58 kDa and 87 kDa, respectively, calculated on the basis of SDS-page, and were both collected in the insoluble fraction. For purification strategy, the two over-expressed transporters were washed with a buffer containing sarkosyl and urea and applied on different Ni2+-chelating chromatography columns. The protein OCTN1 was eluted with a buffer containing 50 mM imidazole and 0.1 % Triton X-100, in quantitiy of 3 mg per litre of cell culture, and then incubated with -mercaptoethanol, to strongly reduce the disulphide bonds; by this way, a different migration on SDS-page to exact molecular weight was observed. The protein OCTN2, previous to be applied on a chromatography column, was treated with DTE and then eluted in the same buffer used for OCTN1 but at different pH. About 0.2 mg of protein per liter of cell culture were obtained. OCTN2 was then, incubated with thrombin 0.1U/l at 25°C and after 4 hours we observed the separation of hOCTN2 and GST fusion tag on SDS-page.