[55] Purification of F1F0 H+-ATPase from Escherichia coli☆

Publisher Summary This chapter summarizes a method for purification of the F 1 F 0 H + -ATPase of Escherichia coli to homogeneity. The chapter also describes the modifications of the basic method for purification of F 1 F 0 from membranes where the complex is overproduced by 6- to 8-fold. Successful application of this method may depend upon use of an equivalent strain, growth conditions, and the method of membrane preparation. The major advantage of this method is the consistent purity of product. The ratio of subunits in the final product is constant from preparation to preparation. The drawbacks of the methods described are the low yields and inherent difficulty in increasing the scale of the procedure. The polypeptide is a major impurity of the slower sedimenting sucrose density gradient fractions that one normally discards, and can easily be mistaken for the chi (χ) subunit.