PUBLICATION
High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli
- Authors
- Senga, Y., Akizuki, K., Katayama, S., Shigeri, Y., Kameshita, I., Ishida, A., Sueyoshi, N.
- ID
- ZDB-PUB-160522-4
- Date
- 2016
- Source
- Biochemical and Biophysical Research Communications 475(3): 277-82 (Journal)
- Registered Authors
- Keywords
- Autonomous, Catalytic fragment, One-step purification, Overexpression, Thermal stability, Truncation
- MeSH Terms
-
- Substrate Specificity
- Zebrafish/genetics
- Zebrafish/metabolism*
- Animals
- Enzyme Stability
- Escherichia coli/genetics
- Zebrafish Proteins/chemistry
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism*
- Cloning, Molecular
- Phosphorylation
- Cyclic AMP-Dependent Protein Kinases/metabolism
- Calcium-Calmodulin-Dependent Protein Kinase Type 1/chemistry
- Calcium-Calmodulin-Dependent Protein Kinase Type 1/genetics
- Calcium-Calmodulin-Dependent Protein Kinase Type 1/metabolism*
- Catalytic Domain
- PubMed
- 27207832 Full text @ Biochem. Biophys. Res. Commun.
Citation
Senga, Y., Akizuki, K., Katayama, S., Shigeri, Y., Kameshita, I., Ishida, A., Sueyoshi, N. (2016) High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli. Biochemical and Biophysical Research Communications. 475(3):277-82.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping