Strategies for rapid purification of catalytic subunits of cAMP-dependent protein kinase

Knowledge of the crystal structure of the catalytic subunit (C) of cAMP-dependent protein kinase provided for the first time a molecular basis for probing function by site-directed mutagenesis. The purification of mutant C-subunits, however, presented new and unanticipated challenges due to instability, insolubility, and underphosphorylation of the altered proteins, To overcome these barriers, a rapid and efficient method for purifying recombinantly expressed C-subunits was developed. Purification to near homogeneity is achieved in less than 5 h, The procedure is based on colysis of bacteria that overexpress the C-subunit with bacteria that overexpress a poly-His-tagged mutant of the type Il regulatory subunit H(6)R(II)(R213K). This mutant R-subunit with an altered cAMP binding site A forms holoenzyme rapidly in bacterial extracts, and the K-a (cAMP) for the resulting holoenzyme, 27-37 mu M, is nearly 50-fold increased compared to holoenzyme formed with wild-type R(II). Thus, after batchwise immobilizing the holoenzyme on Ni2+-resin, the free C-subunit can be directly eluted batchwise with high concentrations of cAMP. The method is described for the purification of wild-type C, with yields of approximately 5 mg/liter, In addition, a mutant subunit, C[G52S], which is defective in ATP binding and could not be isolated using previously described methods, was purified with equal efficiency. (C) 1997 Academic Press.