title: Expression and purification of a convenient Ca2+-calmodulin-dependent protein kinase II GST-fusion substrate.
abstract: Abundant and convenient protein substrates are extremely useful tools for studying protein kinases. However, few such substrates exist for alpha-Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and those that are available are generally small and expensive peptides that are cumbersome to use. The GST-fusion expression system was used to express a 10 amino acid substrate of CaMKII PLRRTLSVAA in bacteria. Using glutathione-agarose affinity chromatography, we obtained milligram quantities of the highly purified recombinant GST-fusion protein. The GST-fusion protein was tested for its efficacy and specificity as a substrate for CaMKII in phosphorylation assays using recombinant enzyme and radiolabeled [gamma-32P]ATP. The reaction products of these phosphorylation assays were resolved by electrophoresis in SDS-polyacrylamide gels and quantified by phosphoimage analysis. It was found that compared to a phosphorylation-null substrate, GST-PLRRTLAVAA, in which the phosphorylated target serine residue was mutated to an alanine, the GST-PLRRTLSVAA substrate was phosphorylated by CaMKII with an apparent K(m) of 18 microM, indicating that the latter is a highly effective substrate for this enzyme.
authors.affiliation: Medical Biotechnology Center, University of Maryland Biotechnology Institute, Room N352, 725 West Lombard Street, Baltimore, MD 21201, USA.
authors.forename: Greta
authors.initials: G
authors.lastname: Miroy
authors.forename: Mervyn J
authors.initials: MJ
authors.lastname: Monteiro
mesh_terms: D000595:Amino Acid Sequence; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006006:Glycogen Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D006006:Glycogen Synthase; D005982:Glutathione Transferase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases