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]α-methyl d-glucopyranoside uptake. We observed a rapid (4–12 h) expression of saturable ( K t = 342 μm) [ 14C]α-methyl d-glucopyranoside uptake following transfection, with substrate and inhibitor sensitivities of the native carrier, including Na + and temperature dependence and appropriate phloridzin sensitivity ( K I = 9.1 μm). The time-dependent increase in α-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 demonstrate 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]γ-aminobutyric acid uptake, we were albe to detect expression from transporter bearing plasmids diluted as much as 10,000-fold by nonspecific plasmid DNA. Thus, these findings document the feasibility of using the vaccinia/T7 expression system for the functional screening of plasmid libraries for membrane transporter cDNA clones and identify several assay variables pertinent to achieving increased sensitivity.