Vaccinia-T7 RNA polymerase expression system: evaluation for the expression cloning of plasma membrane transporters.

Blakely RD, Clark JA, Rudnick G, Amara SG
Anal Biochem. 1991 194 (2): 302-8

PMID: 1862934 · DOI:10.1016/0003-2697(91)90233-j

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)

MeSH Terms (17)

Animals Base Sequence Carrier Proteins Cell Membrane Cloning, Molecular DNA DNA-Directed RNA Polymerases Genetic Vectors HeLa Cells Humans Kinetics Molecular Sequence Data Monosaccharide Transport Proteins Rabbits Transfection Vaccinia virus Viral Proteins

Connections (2)

This publication is referenced by other Labnodes entities:

Links