title: High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli.
abstract: We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation.
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Senga
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Akizuki
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan; Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga 525-8577, Japan.
authors.forename: Syouichi
authors.initials: S
authors.lastname: Katayama
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shigeri
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors.forename: Isamu
authors.initials: I
authors.lastname: Kameshita
authors.affiliation: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. Electronic address: aishida@hiroshima-u.ac.jp.
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Ishida
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan. Electronic address: sueyoshi@ag.kagawa-u.ac.jp.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sueyoshi
mesh_terms: D000818:Animals; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D010766:Phosphorylation; D013379:Substrate Specificity; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029961:Zebrafish Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1
keywords: Autonomous; Catalytic fragment; One-step purification; Overexpression; Thermal stability; Truncation