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

Abstract

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)

• Authors: R D Blakely, J A Clark, G Rudnick, S G Amara
• Journal: Analytical biochemistry (Anal Biochem) Vol. 194 Issue 2 Pg. 302-8 (May 01 1991) ISSN: 0003-2697 [Print] United States
• PMID: 1862934 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Carrier Proteins
• Monosaccharide Transport Proteins
• Viral Proteins
• DNA
• bacteriophage T7 RNA polymerase
• DNA-Directed RNA Polymerases

Topics

• 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