Vaccinia-T7 RNA polymerase expression system: evaluation for the expression cloning of plasma membrane transporters.
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)
Blakely
R D
RD
Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510.
Clark
J A
JA
Rudnick
G
G
Amara
S G
SG
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics*; D014764:Viral Proteins
D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases