dc.description.abstract | Amino 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 |