APPENDIX B
 DEVELOPING PROTOCOL FOR PURIFYING F1Fo ATP SYNTHASE

 Purification of Enzyme Complexes Incorporated with b Subunit Heterodimers

 The purification procedure described below has been attempted one time; therefore

a detailed account of the current protocol is given here. Once the particulars of the

procedure are optimized, the same protocol will be applied to all the F1Fo ATP synthases

incorporated with different combinations of heterodimeric mutant b subunits. For the

sake of simplicity, the different b subunits are simply referred to as bV5 and bhis

Culture

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

subunits will be prepared essentially as described previously and optimized for large-

scale membrane preparations (199, 297). One of the advantages of studying a bacterial

enzyme is the practically unlimited amount of bacteria that may be grown and harvested.

The following can be scaled up or down to meet the experimental needs. The bacteria

will grown by inoculating 20 mL starter culture, grown overnight, into a 2 L Erlenmeyer

flask holding 1 L LBG supplemented with both ampicillin (Ap) (100 Clg/mL) and

chloramphenicol (Cm) (25 Clg/mL). Similarly, 1 mL of the starter culture will be

inoculated into a nephalo flask containing 50 mL LBG (Ap and/or Cm) to monitor

growth. For the purposes of the large-scale purification, a total of 10 L LBG-Ap-Cm,

contained in 10 flasks, will be grown. The bacteria will be grown at 37 oC in a New

Brunswick Scientific incubator shaker (220 rpm) and the turbidity monitored using a

Klett-Summerson photoelectric colorimeter. IPTG (40 CIM) will be added when the


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