An expression system for mammalian amino acid transporters using a stably maintained episomal vector

James C. Matthews, Ara M. Aslanian, Kelly K. McDonald, Wenbo Yang, Marc S. Malandro, Donald A. Novak, Michael S. Kilberg

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

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.

Original languageEnglish
Pages (from-to)208-214
Number of pages7
JournalAnalytical Biochemistry
Volume254
Issue number2
DOIs
StatePublished - Dec 15 1997

Bibliographical note

Funding Information:
This research was supported by the National Institutes of Health, the National Institute of Child Health and Human Resources (HD-29934 to D.A.N.), the Institute for Diabetes, Digestive, and Kidney Diseases (DK-28374 to M.S.K.), and an Institutional National Research Service Award (DK07667 to J.C.M.).

Funding

This research was supported by the National Institutes of Health, the National Institute of Child Health and Human Resources (HD-29934 to D.A.N.), the Institute for Diabetes, Digestive, and Kidney Diseases (DK-28374 to M.S.K.), and an Institutional National Research Service Award (DK07667 to J.C.M.).

FundersFunder number
Institute for Diabetes, Digestive, and Kidney DiseasesDK07667, DK-28374
National Institutes of Health (NIH)
NIH National Institute of Child Health and Human Development National Center for Medical Rehabilitation Research
Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentR01HD029934
Eunice Kennedy Shriver National Institute of Child Health and Human Development

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology
    • Cell Biology

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