intrinsic added start codon, which is in the Sphl recognition sequence, to prevent a

missense mutation. The mutagenic oligonucleotide was designed to accomplish three

tasks in one reaction: 1) mutate the "ATG" found in the Sphl recognition sequence, 2)

add a new Shine Dalgarno sequence in a favorable position from the true start codon, and

3) mutate the "GTG" start codon to a more favorable "ATG" start site (Figure 2-1). The

resulting 3.7 kb plasmid, pTAM40, expressed bA7-HA and b+7-his from the same promoter

and included the ampicillin resistance gene and the pUC 18 origin of replication (Figure

2-2C). In similar constructions, plasmids pTAM41 (bwt-HA and b+7-his) and pTAM42 (bwt-

HA and bwt-his) were created (Table 2-1). Throughout this dissertation, the insertion or

deletion and the epitope tag are indicated after the plasmid name for clarity, for example

plasmid pTAM35 (b+7-his). Each plasmid and the control plasmids pKAM14 (b) and

pBR322 were expressed in the E. coli cell line KM2 (Ab) for study, so that the only b

subunits in the cells were the product of the plasmid genes.

 The two plasmid expression system successfully allowed expression of various

combinations of histidine tagged and V5-tagged b subunits in the same cell (Figure 2-7).

Appropriate antibiotics were added to the growth medium, and in the case of the

coexpressed plasmids, both ampicillin and chloramphenicol were added to select for cells

expressing both plasmids.

Crude Preparative Procedures

 Inverted membrane vesicles from KM2(Ab) strains expressing the desired b subunit

epitope tagged F1Fo ATP synthase complex were prepared for activity assays, Ni-resin

purification and Western Blot analysis. Unless otherwise noted, all reagents, rotors and

materials were kept at 4 oC. The membrane preparations were prepared by inoculating a