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 language | English |
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Pages (from-to) | 208-214 |
Number of pages | 7 |
Journal | Analytical Biochemistry |
Volume | 254 |
Issue number | 2 |
DOIs | |
State | Published - 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.).
Funders | Funder number |
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Institute for Diabetes, Digestive, and Kidney Diseases | DK07667, 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 Development | R01HD029934 |
Eunice Kennedy Shriver National Institute of Child Health and Human Development |
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology