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.
['Kameshita|Isamu|I|', 'Yamashita|Sho|S|', 'Katayama|Syouichi|S|', 'Senga|Yukako|Y|', 'Sueyoshi|Noriyuki|N|']
D000595:Amino Acid Sequence D000818:Animals D015153:Blotting, Western D002417:Cattle D057075:Enzyme Assays D008969:Molecular Sequence Data D004676:Myelin Basic Protein D010766:Phosphorylation D020033:Protein Isoforms D011494:Protein Kinases D011993:Recombinant Fusion Proteins D015203:Reproducibility of Results D017386:Sequence Homology, Amino Acid D013379:Substrate Specificity
protein kinase myelin basic protein phosphorylation In vitro kinase assay kinase substrate