Очистка белка и создание поликлональных антител изофермента креатинкиназы головного мозга

Here we present a purification report on recombinant human brain-type creatine kinase (CKBB). CKBB is the enzyme responsible for energy homeostasis in cell milieu, which reversibly catalyzing the reaction of ATP and ADP synthesis. CKBB is shown to be the crucial enzyme participating in the development of neurodegenerative disorders. During this investigation we used pET-17b plasmid for construction non tagged CKBB protein and pET-14b with attached His-tag sequence. Purified proteins were subjected to SDS-Page electrophoresis to confirm correct protein masses. We demonstrate successful retrieval of a clean purified brain-type creatine kinase expressed in BL21 cell. Our results were supported by western blot analysis with precipitated primarily antibodies raised against recombinant CKBB in rabbit. References 1. Burklen S., Schlattner U., Homayouni R., Gough K., Rak M., Szeghalmi A., and Wallimann T. / J. Biomed. Biotechnol. 2006. V.2006. P.1-11. 2. Aksenov M.Y., Aksenova M.V., Butterfield D.A., Geddes J.W., and Markesbery W.R. / Neuroscience.2001. V.103.P. 373-383. 3. Wu A.H. / Clin Chem. 1989. V.35. No. 1. P. 7-13. 4. Gruemer H.D., Prior T. / Clin. Chim. Acta. 1987. V.162. No. 1. P. 1-18. 5. Chang E.J., Ha J., Oerlemans F., Lee Y.J., Lee Y.J., Lee S.W., Ryu H.J., Kim H.J., Choi J.Y., Kim H.M., Shin C.S., Park Y.K., Tanaka S., Weiringa B., Lee Z.H., Kim H.H. / Nature Med. 2008. V.14. No. 9. P.966-972. 6. Wallimann T., Wyss M., Brdiczka D., Nicolay K., and Eppenberger H.M./ Biochem. J. 1992.V. 281.P. 21-40. 7. Li X.H., Chen Z., Gao Y.S., Yan Y.B., Zhang F., Meng F.G., Zhou H.M. / Int. J. Biol. Macromol. 2011. V. 48. No.2. P. 239-242. 8. Wang P.F., McLeish M.J., Kneen M.M., Lee G., and Kenyon G.L. / Biochemistry. 2001. V. 40. P. 11698-11705. 9. Bong S.M., Moon J.H., Nam K.H., Lee K.S., Chi Y.M., and Hwang K.Y./ FEBS Lett. 2008. V. 582.P. 3959-3965. 10. Chen L.H., Babbitt P.C., Vesquez J.R., West B.L., Kenyon G.L. / J. Biol. Chem. 1991. V. 266. No.18. P. 12053-12057. 11. Chen L.H., White C.B., Babbit P.C., McLeish M.J., Kenyon G.L. / J. Biol. Chem. 2000. V. 19. No.1. P. 59-66. 12. Sunahara Y., Uchida K., Tanaka T., Matsukawa H., Inagaki M., Matuo Y. / Clin. Chem. 2001. V. 47. No. 3. P.471-476. 13. Rakhmetov A., Lee S. P., Ostapchenko L., Chae H. Z. / Vistnyk of KNU. 2013. V. 2(64). P. 58-61. 14. Eppenberger H.M., Dawson D.M., Kaplan N.O. / J. Biol. Chem. 1967. V. 242. No.2. P. 204-209. 15. Friedhoff A.J., Lerner M.H. / Life Sci. 1977. V. 20(5). P. 867-873. 16. Xu C.J., Klunk W.E., Kanfer J.N., Xiong Q., Miller G., Pottegrew J.W. / J Biol Chem. 1996. V. 271(23). P. 13435-13440. 17. David S., Shoemaker M., Haley B.E. / Brain Res Mol. 1998. V. 54(2). P. 276-287. 18. White K.C., Babbitt P.C., Buechter D.D., Kenyon G.L. / J. Protein. Chem. 1992. V. 11. No.5. P 489-494. 19. Eder M., Schlattner U., Becker A., Wallimann T., Kabsch W., Fritz-Wolf K. / Protein Sci. 1999. V. 8. No.11. P. 2258-2269. 20. Crowe J., Dobeli H., Gentz R., Hochuli E., Stuber D., Henco K. / Methods Mol. Biol. 1994. V. 31. P. 371-87.