A method for the purification of cAMP-dependent protein kinase using immunoaffinity chromatography

A rapid and efficient method for purifying cAMP-dependent protein kinase (PKA) holoenzyme based on immunoaffinity chromatography was developed. The affinity column was prepared by coupling a polyclonal antibody raised against the PKA regulatory subunit to NHS-activated Sepharose. The holoenzyme purified by this procedure from the bivalve mollusk Mytilus galloprovincialis was shown to be fully active as judged by (1) its cAMP-binding activity, (2) its cAMP-dependent protein kinase activity, and (3) its autophosphorylation ability. Moreover, together with both regulatory and catalytic subunits, which constitute the PKA holoenzyme, a protein with a molecular mass of approximately 200 kDa was copurified, and results from gel-filtration chromatography showed that it was associated with a fraction of PKA. Therefore, this immunoaffinity purification technique could also be useful to isolate such proteins as interact with PKA in vivo.