title: An expression system for mammalian amino acid transporters using a stably maintained episomal vector.
abstract: Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and X-AG, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNA-transfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein-Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.4x) and EAAC1 (39x) transport activity than does the vaccinia/HeLa expression system (2.6x and 22x, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X-AG, or A; and (iii) the endogenous transport velocities of Systems ASC and X-AG in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA-1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville 32610, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Matthews
authors.forename: A M
authors.initials: AM
authors.lastname: Aslanian
authors.forename: K K
authors.initials: KK
authors.lastname: McDonald
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.forename: M S
authors.initials: MS
authors.lastname: Malandro
authors.forename: D A
authors.initials: DA
authors.lastname: Novak
authors.forename: M S
authors.initials: MS
authors.lastname: Kilberg
mesh_terms: D027322:Amino Acid Transport System X-AG; D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics*; D050580:Excitatory Amino Acid Transporter 3; D015723:Gene Library; D050491:Glutamate Plasma Membrane Transport Proteins; D006624:Hippocampus; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D014162:Transfection / Q000235:genetics*
chemical_list: D027322:Amino Acid Transport System X-AG; D026905:Amino Acid Transport Systems; D000596:Amino Acids; D002352:Carrier Proteins; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D012333:RNA, Messenger; C493400:SLC1A1 protein, human; C493398:Slc1a1 protein, rat; D027981:Symporters; D012964:Sodium