Abstract
Calpain, the Ca2+-dependent cysteine endopeptidase, and its endogenous inhibitory protein calpastatin are distributed in animal cells and are involvedin Ca2+-signalling pathway. Using baculovirus expression system, human µ-calpain with active-site-mutation and calpastatin are expressed and purified. The large subunit of the mutant µ-calpain was processed by the wildtype calpain, supporting the intermolecular cleavage mechanism of procalpain during activation. The mutant µ-calpain had no proteolytic activity, but retained the high ordered structure of wild type calpain. The recombinant calpastatin had inhibitory activity for µ- and m-calpain.
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References
Suzuki, K., Imajoh S., Emori Y., Kawasaki H., Minami, Y. and Ohno S. (1988) Regulation of activity of calcium activated neutral protease, Adv.Enzyme Regul., 27, 153–169.
Mellgren. R.L. and Murachi T. (Eds.), (1990) Intracellular calcium-dependent proteolysis., CRCPress, Boca Raton, FL, U.S.A.
Croall D.E., and DeMartino G.N. (1991) Calcium-activated neutral protease (calpain) system: structure, function, and regulation, Physiological Reviews, 71, 813–847.
Murachi, T. (1983) Calpain and calpastatin, Trends in Biochem. Sci., 8, 167–169.
Ariyoshi, E., Shiba, M., Sakon, Kambayashi J., Yoshida K., Kawashima S., and Mori T. (1993) Translation of human platelet calpain-I, Biochem. & Mol. Biol. Int., 30, 63–72.
Emori, Y. and Saigo, K. (1994) Calpain localization changes in coordination with actin-related cytoskeletal changes during early embryonic development of Drosophila, J. Biol. Chem., 269, 25137–25142.
Saito K., Elce J.S., Hamos J.E., and Nixon R.A. (1993) Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration. Proc. Natl. Acad. Sci. U.S.A., 90, 2628–2632.
Parkes, (1986) in Preteinase Inhibitors (A.J., Barrett, and G., Salvesen eds.) Elsevier Science Publishers, B.V., Amsterdam, 571–587.
Maki, M., Takano, E., Osawa, T., Ooi, T., Murachi, T., and Hatanaka, M. (1988) Analysis of structure and function relationship of pig calpastatin by expression of mutated cDNAs in Esherichia coli.. J. Biol. Chem., 263, 10254–10261.
Baculovirus Expression Protocols, ed. by Pichardson C.D., (1995) Methods in Molecular Biology, Humana Press, Totowa, New Jersey.
Hitomi, K., Yokoyama, A. and Maki, M.(1998) Expression of biologically active human calpastatin in baculovirus-infected insect cells and in Ecsherichia coli. Biochem. Biosci. Biotech., 62, 136–141.
Hitomi, K., Utiyama, Y., Ohkubo, I., Kunimatsu, M., Sasaki, M., and Maki, M. (1998) Purification and characterization of the active-site-mutated recombinant human mu-calpain in baculovirus-infected Insect cell. Biochem. Biophys. Res. Commun., 246, 681–685.
Meyer S.L., Bozyczko-coyne D., Mallya, S.K., Spais C.M., Bihovsky R., Kawooya J.K., Lang D.M., Scott R.W., and Siman R., (1996) Biologically active monomeric and heterodimeric recmbinant human calpain I produced using the baculovirus expression system. Biochem. J., 314, 511–519.
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© 1999 Kluwer Academic Publishers
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Hitomi, K., Maki, M. (1999). Expression and Characterization of Human Calpain and Calpastatin Using Baculovirus System. In: Kitagawa, Y., Matsuda, T., Iijima, S. (eds) Animal Cell Technology: Basic & Applied Aspects., vol 1. Springer, Dordrecht. https://doi.org/10.1007/0-306-46865-4_45
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DOI: https://doi.org/10.1007/0-306-46865-4_45
Publisher Name: Springer, Dordrecht
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