High-level expression using baculovirus, purification, and characterization of a monomeric form of type II calmodulin-dependent protein kinase.
The type II calmodulin-dependent protein kinase is an oligomer existing in multiple isozymic forms. To facilitate investigations of the regulatory mechanisms of this complex enzyme, we have constructed a truncated, calmodulin-dependent monomer of the alpha subunit. The N-terminal enzyme fragment (alpha 315) was expressed at high levels in a baculovirus/insect cell expression system. The recombinant protein was purified chromatographically using DEAE-cellulose, calmodulin-Sepharose, and AffiGel blue, yielding 4 mg of kinase from 1.5 x 10(8) cells in 4 h. Characterization of the truncated kinase indicated that it is a monomer and that interactions of alpha 315 with calmodulin and substrates are indistinguishable from those observed for purified holoenzyme from rat brain. These results indicate that the baculovirus/insect cell expression system is well suited for producing alpha 315, a structurally simplified model of the type II calmodulin-dependent protein kinase.
Takeuchi-Suzuki
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E
Department of Chemistry, Ohio State University, Columbus 43210.
Tanaka
T
T
Hink
W F
WF
King
M M
MM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002478:Cells, Cultured; D005822:Genetic Vectors; D009036:Moths; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011494:Protein Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases