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Heterologous over-expression of the human amino acid transportes LAT1-CD98 and ASCT2

dc.contributor.authorPingitore, Piero
dc.contributor.authorPochinico, Lorena
dc.contributor.authorCerra, Maria Carmela
dc.date.accessioned2016-03-03T11:51:54Z
dc.date.available2016-03-03T11:51:54Z
dc.date.issued2013-12-02
dc.identifier.urihttp://hdl.handle.net/10955/792
dc.identifier.urihttp://dx.doi.org/10.13126/UNICAL.IT/DOTTORATI/792
dc.descriptionDottorato di Ricerca in Biologia Animale Ciclo XXVI, a.a.2013en_US
dc.description.abstractAmino acid transport across the plasma membrane in mammalian cells is mediated by different transport systems such as the Na+-dependent systems A, ASC and N and the Na+- independent system L. Very interestingly some of these transporters such as ASCT2 and LAT1 are over-expressed in many tumors. Cancer cells, in fact, display enhanced need for amino acids and altered amino acid metabolism. Thus, structural and functional studies of these transporters are very important not only for characterization but also for applications in human therapy. Over-expression of the transport proteins is the starting point for obtaining purified transporters. Bacterial and/or yeast cell systems have been employed for this purpose, so far. LAT1 (SLC7A5) belongs to the system L which catalyze the transport of branched chain and aromatic amino acids. LAT1 is an heterodimer and its counterpart, CD98 (SLC3A2) protein, is probably involved in substrate recognition and membrane localization. Bacterial over-expression of the hLAT1 transporter has been performed using a screening strategy of E. coli strains transformed with several plasmid constructs. The best expression of the hLAT1 protein was achieved after cloning of the cDNA into pH6EX3 vector and transformation of Rosetta(DE3)pLysS cells. The hLAT1 protein was purified by Ni2+- chelating chromatography with a yield of about 3.5 mg/L. The cDNA coding for hCD98 was cloned in the pGEX-4T1 vector containing a N-terminal GST tag. Protein expression was obtained using the same bacterial strain above described. Differently from hLAT1, the GST-CD98 protein was soluble. hCD98 was obtained after thrombin treatment and separation by size exclusion chromatography, with a yield of 2 mg/L. ASCT2 (SLC1A5) belongs to the system ASC and has high affinity for Ala, Ser, Cys, Gln and Asn. E. coli revealed not suitable for expressing this protein. Thus, a different approach using P. pastoris was performed to produce the recombinant hASCT2 protein. After codon optimization for P. pastoris, the hASCT2 cDNA was cloned in the pPICZB expression vector carrying a C-terminal 6His-tag. For large protein production, the recombinant P. pastoris strain was grown in fermentors. The recombinant proteins was mainly localized to the membrane. After purification using Ni2+-NTA resin a yield of at least 10 mg/L was obtained. The procedure described can be now used for producing the three proteins in appropriate amounts for crystallization trials and functional studies.en_US
dc.description.sponsorshipUniversità della calabriaen_US
dc.language.isoenen_US
dc.relation.ispartofseriesBIO/11;
dc.subjectBiologia molecolareen_US
dc.subjectAmminoacidien_US
dc.subjectProteine di trasportoen_US
dc.titleHeterologous over-expression of the human amino acid transportes LAT1-CD98 and ASCT2en_US
dc.typeThesisen_US


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