The C-terminus of CaMKII is truncated when expressed in E. coli.
The neuronal enzyme Calcium/calmodulin dependent protein kinase type II (CaMKII) is a key molecule in biochemical events necessary for learning and memory. The alpha-subunit of CaMKII expressed in E. coli as well as in insect cells shows similar catalytic behavior [Praseeda, M., Pradeep, K. K., Krupa, A., Sri Krishna, S., Leena, S., Rajeev Kumar, R., John Cheriyan, Mayadevi, M., Srinivasan, N., and Omkumar, R. V. (2003) Biochem. J. In Press]. The association domain of the enzyme has been crystallized in its native multimeric form after expression in E. coli [Hoelz, A., Nairn, A. C. and Kuriyan, J. (2003) Molecular Cell 11, 1241]. However a major truncation product accompanies the full-length protein when expressed in E. coli. We show by epitope labeling and immunoblotting that the truncation occurs at the C-terminal half of the protein so that the N-terminal catalytic domain is complete in the truncated product. This supports the use of the preparation of alpha-CaMKII expressed in E. coli for studies on functions of the catalytic site. Our data will also be helpful in designing modified prokaryotic expression systems for CaMKII devoid of the trun-cation product, which are easier to use compared to the insect cell system.
Praseeda
M
M
Rajiv Gandhi Centre for Biotechnology, Thycaud P. O., Thiruvananthapuram, Kerala-695014, India.
Beena
Mary K
MK
Asha
Sarah John
SJ
Omkumar
R V
RV
D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000939:Epitopes; D011993:Recombinant Fusion Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases