TandeMBP: generation of a unique protein substrate for protein kinase assays.
Myelin basic protein (MBP) is one of the major components of central nervous system myelin and has multiple sites for protein phosphorylation. Therefore, it has been widely used as a substrate for in vitro assays of various protein kinases. In this study, to obtain more efficient substrates for protein kinase assays than commercially available MBP from bovine brain, we produced various recombinant MBPs using Escherichia coli expression systems. Three splice isoforms of mouse MBP were expressed in E. coli and successfully purified using a new protocol consisting of HCl extraction, urea treatment and affinity purification with HiTrap Chelating HP column. The recombinant MBP isoforms thus obtained served as more efficient substrates for protein kinases than MBP isolated from bovine brain. To generate an even better substrate for protein kinase assays, we produced a hybrid protein composed of two different MBP isoforms connected in tandem, designated TandeMBP. TandeMBP was readily expressed in E. coli and could be purified by the newly developed simple procedure. TandeMBP was phosphorylated by various Ser/Thr protein kinases more efficiently than the other MBP isoforms. Taken together, TandeMBP will become a powerful tool for in vitro assays to analyse various protein kinase activities.
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan kamesita@ag.kagawa-u.ac.jp.
Isamu
I
Kameshita
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
Sho
S
Yamashita
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
Syouichi
S
Katayama
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
Yukako
Y
Senga
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
Noriyuki
N
Sueyoshi
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D057075:Enzyme Assays / Q000379:methods; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004676:Myelin Basic Protein; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases
In vitro kinase assay; kinase substrate; myelin basic protein; phosphorylation; protein kinase