Journal Article

TandeMBP: generation of a unique protein substrate for protein kinase assays

Isamu Kameshita, Sho Yamashita, Syouichi Katayama, Yukako Senga and Noriyuki Sueyoshi

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 156, issue 3, pages 147-154
Published in print September 2014 | ISSN: 0021-924X
Published online April 2014 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/jb/mvu025
TandeMBP: generation of a unique protein substrate for protein kinase assays

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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.

Keywords: In vitro kinase assay; kinase substrate; myelin basic protein; phosphorylation; protein kinase

Journal Article.  4723 words.  Illustrated.

Subjects: Biochemistry

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