Espressione di sistemi di trasporto umani di membrana plasmatica in E.coli
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Amelio, Linda
Cerra, Maria Carmela
Pochini, Lorena
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Dottorato di Ricerca in Biologia Animale, XXIII Ciclo,a.a. 2009-2010; 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.; Università della CalabriaSoggetto
Membrane plasmatiche
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BIO/10;