| Vaccinia-T7 RNA polymerase expression system: evaluation for the expression cloning of plasma membrane transporters. | |
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MedLine Citation:
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PMID: 1862934 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The vaccinia/T7 transient expression system, which results in rapid, high-level expression of proteins encoded by plasmids bearing T7 promoters, provides a powerful strategy for the expression cloning of membrane transporters. To test the feasibility of this approach, we introduced the rabbit Na+/glucose transporter by liposome-mediated transfection into vaccinia infected HeLa cells and determined the characteristics and sensitivity of induced [14C]alpha-methyl D-glucopyranoside uptake. We observed a rapid (4-12 h) expression of saturable (Kt = 342 microM) [14C]alpha-methyl D-glucopyranoside uptake following transfection, with substrate and inhibitor sensitivities of the native carrier, including Na+ and temperature dependence and appropriate phloridzin sensitivity (KI = 9.1 microM). The time-dependent increase in alpha-methyl D-glucopyranoside uptake coincided with a decline in endogenous Na+/D-aspartate transport. Maximal levels of expression achieved were nearly 10-fold higher than that reported for transient expression of Na+/glucose transporters in the COS cell system. Rate and dilution estimates demonstrates a sensitivity of detection of single clones diluted several thousand fold by nonspecific plasmid DNA. A further 3-fold increase in transport sensitivity was achieved after transfection of plasmid constructs bearing additional 5'-T7 stem-loop and 3'-T7 termination signals. When cell lines with low endogenous transport were coupled with substrates of high specific activity, as with measurements of induced [3H]gamma-aminobutyric acid uptake, we were able to detect expression from transporter bearing plasmids diluted as much as 10,000-fold by non-specific plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS) |
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Authors:
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R D Blakely; J A Clark; G Rudnick; S G Amara |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Analytical biochemistry Volume: 194 ISSN: 0003-2697 ISO Abbreviation: Anal. Biochem. Publication Date: 1991 May |
Date Detail:
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Created Date: 1991-08-30 Completed Date: 1991-08-30 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 0370535 Medline TA: Anal Biochem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 302-8 Citation Subset: IM |
Affiliation:
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Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Base Sequence Carrier Proteins / genetics*, metabolism Cell Membrane / metabolism Cloning, Molecular / methods* DNA DNA-Directed RNA Polymerases / genetics* Genetic Vectors* Hela Cells Humans Kinetics Molecular Sequence Data Monosaccharide Transport Proteins / genetics, metabolism Rabbits Transfection Vaccinia virus / genetics* Viral Proteins |
| Grant Support | |
ID/Acronym/Agency:
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5 T32 GM07324-14/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Carrier Proteins; 0/Monosaccharide Transport Proteins; 0/Viral Proteins; 9007-49-2/DNA; EC 2.7.7.-/bacteriophage T7 RNA polymerase; EC 2.7.7.6/DNA-Directed RNA Polymerases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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