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Cloning, Expression, Purification and Refolding of Microtubule Affinity-Regulating Kinase 4 Expressed in Escherichia coli

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Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a member of the family Ser/Thr kinase and involved in numerous biological functions including microtubule bundle formation, nervous system development, positive regulation of programmed cell death, cell cycle control, cell polarity determination, cell shape alterations, cell division etc. For various biophysical and structural studies, we need this protein in adequate quantity. In this paper, we report a novel cloning strategy for MARK4. We have cloned MARK4 catalytic domain including 59 N-terminal extra residues with unknown function and catalytic domain alone in PQE30 vector. The recombinant MARK4 was expressed in the inclusion bodies in M15 cells. The inclusion bodies were solubilized effectively with 1.5 % N-lauroylsarcosine in alkaline buffer and subsequently purified using Ni–NTA affinity chromatography in a single step with high purity and good concentration. Purity of protein was checked on sodium dodecyl sulphate–polyacrylamide gel electrophoresis and identified by using mass spectrometry immunoblotting. Refolding of the recombinant protein was validated by ATPase assay. Our purification procedure is quick, simple and produces adequate quantity of proteins with high purity in a limited step.

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  1. Espinosa, L., & Navarro, E. (1998). Cytogenetics and Cell Genetics, 81(3–4), 278–82.

    Article  CAS  Google Scholar 

  2. Naz, F., Anjum, F., Islam, A., et al. (2013). Cell Biochemistry and Biophysics, 67(2), 485–99.

  3. Marx, A., Nugoor, C., Panneerselvam, S., et al. (2010). The FASEB Journal, 24(6), 1637–48.

    Article  CAS  Google Scholar 

  4. Kato, T., Satoh, S., Okabe, H., et al. (2001). Neoplasia, 3(1), 4–9.

    Article  CAS  Google Scholar 

  5. Moroni, R. F., De Biasi, S., Colapietro, P., et al. (2006). Neuroscience, 143(1), 83–94.

    Article  CAS  Google Scholar 

  6. Schneider, A., Laage, R., von Ahsen, O., et al. (2004). Journal of Neurochemistry, 88(5), 1114–26.

    Article  CAS  Google Scholar 

  7. Trinczek, B., Brajenovic, M., Ebneth, A., et al. (2004). The Journal of Biological Chemistry, 279(7), 5915–23.

    Article  CAS  Google Scholar 

  8. Schmitt-Ulms, G., Matenia, D., Drewes, G., et al. (2009). Cell Motility and the Cytoskeleton, 66(8), 661–72.

    Article  CAS  Google Scholar 

  9. Magnani, I., Novielli, C., Bellini, M., et al. (2009). Cellular Oncology, 31(5), 357–70.

    CAS  Google Scholar 

  10. Roversi, G., Pfundt, R., Moroni, R. F., et al. (2006). Oncogene, 25(10), 1571–83.

    Article  CAS  Google Scholar 

  11. Magnani, I., Moroni, R. F., Roversi, G., et al. (2005). Cancer Genetics and Cytogenetics, 161(2), 140–5.

    Article  CAS  Google Scholar 

  12. Beghini, A., Magnani, I., Roversi, G., et al. (2003). Oncogene, 22(17), 2581–91.

    Article  CAS  Google Scholar 

  13. Brajenovic, M., Joberty, G., Kuster, B., et al. (2004). The Journal of Biological Chemistry, 279(13), 12804–11.

    Article  CAS  Google Scholar 

  14. Wojcik, E., Basto, R., Serr, M., et al. (2001). Nature Cell Biology, 3(11), 1001–7.

    Article  CAS  Google Scholar 

  15. Lilie, H., Schwarz, E., & Rudolph, R. (1998). Current Opinion in Biotechnology, 9(5), 497–501.

    Article  CAS  Google Scholar 

  16. Vallejo, L. F., Brokelmann, M., Marten, S., et al. (2002). Journal of Biotechnology, 94(2), 185–94.

    Article  CAS  Google Scholar 

  17. Roder, H., & Shastry, M. R. (1999). Current Opinion in Structural Biology, 9(5), 620–6.

    Article  CAS  Google Scholar 

  18. Clark, E. D. B. (1998). Current Opinion in Biotechnology, 9(2), 157–63.

    Article  Google Scholar 

  19. Sambrook, D. W., Jr. (2001). Molecular cloning: a laboratory manual (3rd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory.

    Google Scholar 

  20. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–54.

    Article  CAS  Google Scholar 

  21. Blancher, C., & Jones, A. (2001). Methods in Molecular Medicine, 57, 145–62.

    CAS  Google Scholar 

  22. Hassan, M. I., Kumar, V., Kashav, T., et al. (2007). Journal of Separation Science, 30(12), 1979–88.

    Article  CAS  Google Scholar 

  23. Hassan, M. I., Kumar, V., Singh, T. P., et al. (2008). Journal of Separation Science, 31(12), 2318–24.

    Article  CAS  Google Scholar 

  24. Kim, S. H. (2003). Protein Expression and Purification, 27(1), 85–9.

    Article  CAS  Google Scholar 

  25. Kurucz, I., Titus, J. A., Jost, C. R., et al. (1995). Molecular Immunology, 32(17–18), 1443–52.

    Article  CAS  Google Scholar 

  26. Grisshammer, R., & Tucker, J. (1997). Protein Expression and Purification, 11(1), 53–60.

    Article  CAS  Google Scholar 

  27. Chu, X., & Li, D. (2003). Protein Expression and Purification, 27(1), 165–70.

    Article  CAS  Google Scholar 

  28. Carrio, M. M., & Villaverde, A. (2002). Journal of Biotechnology, 96(1), 3–12.

    Article  CAS  Google Scholar 

  29. Babu, K. R., Swaminathan, S., Marten, S., et al. (2000). Applied Microbiology and Biotechnology, 53(6), 655–60.

    Article  CAS  Google Scholar 

  30. Misawa, S., & Kumagai, I. (1999). Biopolymers, 51(4), 297–307.

    Article  CAS  Google Scholar 

  31. Shan, J., Baguinon, M., Zheng, L., et al. (2003). Protein Expression and Purification, 27(1), 143–9.

    Article  CAS  Google Scholar 

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FN acknowledges the Council of Scientific and Industrial Research for the award of fellowship. We sincerely thank Harvard Medical School for providing MARK4 cDNA. FA and MIH are thankful to the Department of Science and Technology for funding.

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Correspondence to Md Imtaiyaz Hassan.

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Naz, F., Asad, M., Malhotra, P. et al. Cloning, Expression, Purification and Refolding of Microtubule Affinity-Regulating Kinase 4 Expressed in Escherichia coli . Appl Biochem Biotechnol 172, 2838–2848 (2014).

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