High level expression and preparation of autonomous Ca2+/calmodulin-dependent protein kinase II in Escherichia coli.
The chymotryptic fragment of Ca2+/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active enzyme that phosphorylates a variety of protein substrates in vitro. Although 30K-CaMKII is an often used and powerful tool for protein phosphorylation, the efficient production of catalytically active 30K-CaMKII in Escherichia coli has not yet been successfully realized, probably due to its toxicity in host cells. In this study, we found that a high-level expression of 30K-CaMKII as an insoluble form was attained when the N-terminal 43 amino acid residues of Xenopus CaMKI were fused to the N-terminal end of 30K-CaMKII (CX-30K-CaMKII). The inactive CX-30K-CaMKII thus expressed in E. coli was reactivated by simple denaturation/renaturation processes and purified on a Ni2+-chelating column. The renatured CX-30K-CaMKII exhibited specific activity similar to that of rat brain CaMKII, and phosphorylated various proteins such as histones, myosin light chain, myelin basic protein, and synapsin I, as in case of 30K-CaMKII or purified CaMKII. Thus, CX-30K-CaMKII, an autonomous CaMKII, can be obtained with a simple procedure using E. coli expression system.
Shoju
Hiroyuki
H
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795.
Sueyoshi
Noriyuki
N
Ishida
Atsuhiko
A
Kameshita
Isamu
I
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D020673:Protein Renaturation; D014982:Xenopus laevis
D017871:Calcium-Calmodulin-Dependent Protein Kinases