medium was used as an initial qualitative gauge of enzyme activity in vivo since E. coli

strains lacking F1Fo ATP synthase cannot derive energy from nonfermentable carbon

sources. In each case, the strains expressing the histidine and HA-epitope tagged b

subunits grew comparably to the wild type strain (Table 2-1).



 ori
 Cm


 Ap bA7
 b+7

 pTAM134 pTAM35

 Orl3.2 kb 6.9 kb




 b A7 -H A b+7 -h is


Figure 2-3. Histidine and HA-epitope-tagged b subunit expression system. We employed
 an epitope-tag system in order to determine whether b subunits of unequal
 length can interact to form the dimer in an intact and functional enzyme
 complex. B) Both bA7-HA and b+7-his were expressed together in KM2 (Ab)
 cells using a two-plasmid expression system. Plasmid pTAM34 was designed
 to express high levels of HA-tagged ba7. This plasmid contains the genes
 conferring ampicillin resistance and the pUC18 origin of replication. Plasmid
 pTAM3 5 includes the chloramphenicol resistance gene, the pACYC 184 origin
 of replication and expresses histidine-tagged b 7. In similar constructions,
 plasmids pTAM37 (bwt-his, Cmr) and pTAM36 (bwt-HA, Apr), or pTAM35 (b 7.
 his, Cmr) and pTAM46 (bwt-HA, Apr) were developed for coexpression
 experiments.