Recombinant 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 II regulatory subunit H6RII (R213K). This mutant R-subunit with an altered cAMP binding site A forms holoenzyme rapidly in bacterial extracts, and the Ka (cAMP) for the resulting holoenzyme, 27-37 microM, is nearly 50-fold increased compared to holoenzyme formed with wild-type RII. Thus, after batchwise immobilizing the holoenzyme on Ni(2+)-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.
Hemmer
W
W
Department of Chemistry and Biochemistry, University of California, San Diego 92093-0654, USA.
McGlone
M
M
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D004789:Enzyme Activation; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases