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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Expression, Purification and Transduction of PEP-1-Botulinum Neurotoxin Type A (PEP-1-BoNT/A) into Skin

  • Kim, Dae-Won (Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University) ;
  • Kim, So-Young (Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University) ;
  • An, Jae-Jin (Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University) ;
  • Lee, Sun-Hwa (Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University) ;
  • Jang, Sang-Ho (Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University) ;
  • Won, Moo-Ho (Department of Anatomy, College of Medicine, Hallym University) ;
  • Kang, Tae-Cheon (Department of Anatomy, College of Medicine, Hallym University) ;
  • Chung, Kwang-Hoe (Biobud Co., Ltd) ;
  • Jung, Hyun-Ho (Medy-Tox Inc.) ;
  • Cho, Sung-Woo (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine) ;
  • Choi, Jin-Hi (Research Laboratory of Cell Tech. Korea) ;
  • Park, Jin-Seu (Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University) ;
  • Eum, Won-Sik (Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University) ;
  • Choi, Soo-Young (Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University)
  • Received : 2006.05.14
  • Accepted : 2006.06.05
  • Published : 2006.09.30
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Abstract

Botulinum neurotoxin A (BoNT/A) has been used therapeutically to treat muscular hypercontractions and sudomotor hyperactivity and it has been reported that BoNT/A might have analgesic properties in headache. PEP-1 peptide is a known carrier peptide that delivers fulll-ength native proteins in vitro and in vivo. In this study, a BoNT/A gene were fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-BoNT/A fusion protein. The expressed and purified PEP-1-BoNT/A fusion proteins were efficiently transduced into cells in a time- and dose-dependent manner when added exogenously in a culture medium. In addition, immuno-histochemical analysis revealed that PEP-1-BoNT/A fusion protein efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin. These results suggest that PEP-1-BoNT/A fusion protein provide an efficient strategy for therapeutic delivery in various human diseases related to this protein.

Keywords

References

  1. Arnon, S. A., Schecter, R., Inglesby, T. V., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Fine. A. D., Hauer, J., Layton, M., Lillibridge, S., Osterholm, M. T., O'Toole, T., Parker, G., Perl, T. M., Russell, P. K., Swerdlow, D. L. and Tonat, K. (2001) Botulinum toxin as a biological weapon: medical and public health management. J. Am. Med. Assoc. 285, 1059-1070. https://doi.org/10.1001/jama.285.8.1059
  2. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  3. Carruthers, J. and Carruthers, A. (1998) The adjunctive usage of botulinum toxin. Dermatol. Surg. 24, 1244-1247. https://doi.org/10.1016/S1076-0512(98)00188-5
  4. Center for Disease Control (1998) Botulism in the United States, 1899-1998. Handbook for Epidemiologists, Clinnicans, and Laboratory Workers. Atlanta, Georgia US Department of Health and Human Services, Public Health Service.
  5. Choi, H. S., An, J. J., Kim, S. Y., Lee, S. H., Kim, D. W., Yoo, K. Y., Won, M. H., Kang, T. C., Kwon, H. J., Kang, J. H., Cho, S. W., Kwon, O. S., Park, J., Eum, W. S. and Choi, S. Y. (2006) PEP-1-SOD fusion protein efficiently protects against paraquat-induced dopaminergic neuron in Parkinson's Disease mouse model. Free Radic. Biol. Med. In press.
  6. Choi, S. H., Kim, D. W., Kim, S. Y., An, J. J., Lee, S. H., Sohn, E. J., Hwang, S. I., Won, M. H., Kang, T. C., Kwon, H. J., Kang, J. H., Cho, S. W., Park, J., Eum, W. S. and Choi, S. Y. (2005) Transduced human copper chaperone for Cu, Zn-SOD (PEP-1-CCS) protects against neuronal cell death. Mol. Cells 20, 401-408.
  7. Christopher, G. W., Cieslak, T. J., Pavlin, J. A. and Eitzen Jr, E. M. (1997) Biological warfare. A historical perspective. J. Am. Med. Assoc. 278, 412-417. https://doi.org/10.1001/jama.278.5.412
  8. Cordivari, C., Misra, P. V., Catania, S. and Lees, A. J. (2004) New therapeutic indications for botulinum toxins. Mov. Disord. 19, 157-161. https://doi.org/10.1002/mds.20071
  9. De Maio, M. (2003) Botulinum toxin in associated with other rejuvenation methods. J. Cosmetic Laser Ther. 5, 210-212. https://doi.org/10.1080/14764170310021896
  10. Eum W. S., Choung, I. S., Li, M. Z., Kang, J. H., Kim, D. W., Park, J., Kwon, H. Y. and Choi, S. Y. (2004a) HIV-1 Tat mediated protein transduction of Cu,Zn-superoxide dismutase into pancreatic b cells in vitro and in vivo. Free Radic. Biol. Med. 37, 339-349. https://doi.org/10.1016/j.freeradbiomed.2004.04.036
  11. Eum, W. S., Kim, D. W., Hwang, I. K., Yoo, K. Y., Kang, T. C., Jang, S. H., Choi, H. S., Choi, S. H., Kim, Y. H., Kim, S. Y., Kwon, H. Y., Kang, J. H., Kwon, O. S., Cho, S. W., Lee, K. S., Park, J., Won, M. H. and Choi, S. Y. (2004b) In vivo protein transduction: Biologically active intact PEP-1-superoxide dismutase fusion protein efficiently protects against ischemic insult. Free Radic. Biol. Med. 37, 1656-1669. https://doi.org/10.1016/j.freeradbiomed.2004.07.028
  12. Fawell, S., Seery, J. and Daikh, Y. (1991) Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. USA 91, 664-668.
  13. Ha, K. T., Lee, Y. C., Cho, S. H., Kim, J. K. and Kim, C. H. (2004) Molecular characterization of membrane type and ganglioside-specific sialidase (Neu3) expressed in E. coli. Mol. Cells 17, 267-273.
  14. Hwang, J. H., Yuk, S. H., Lee, J. H., Lyoo, W. S., Ghil, S. H., Lee, S. S., Khang, I. G., Paik, S. Y. and Lee, J. Y. (2004) Differentiation of stem cells isolated from rat smooth muscle. Mol. Cells 17, 57-61.
  15. Kim, C. H. (2003) A Salmonella typhimurium rfaE mutants recovers invasiveness for human epithelial cells when complemented by wild type rfaE (confering biosynthesis of ADP-L-glycero-Dmanno-heptose-containing lipopolysaccharide). Mol. Cells 15, 226-232.
  16. Kim, D. W., Eum, W. S., Jang, S. H., Kim, S. Y., Choi, H. S., Choi, S. H., An, J. J., Lee, S. H., Lee, K. S., Han, K., Kang, T. C., Won, M. H., Kang, J. H., Kwon, O. S., Cho, S. W., Kim, T. Y., Park, J. and Choi, S. Y. (2005) Transduced Tat-SOD fusion protein protects against ischemic brain injury. Mol. Cells 19, 88-96.
  17. Kim, S. Y., An, J. J., Kim, D. W., Choi, S. H., Lee, S. H., Hwang, S. I., Kwon, O. S., Kang, T. C., Won, M. H., Cho, S. W., Park, J., Eum, W. S., Lee, K. S. and Choi, S. Y. (2006) Tat-mediated Protein Transduction of Human Brain Pyridoxine-5-P Oxidase into PC12 Cells. J. Biochem. Mol. Biol. 39, 76-83. https://doi.org/10.5483/BMBRep.2006.39.1.076
  18. Klein, A. W. (2004) The therapeutic potential of botulinum toxin. Dermatol. Surg. 30, 452-455. https://doi.org/10.1111/j.1524-4725.2004.30118.x
  19. Lacy, B. D. and Stevens, R. C. (1999) Sequence homology and structural analysis of the clostridial neurotoxins. J. Mol. Biol. 291, 1091-1104. https://doi.org/10.1006/jmbi.1999.2945
  20. Mauskop, A. (2002) The use of botulinum toxin in the treatment of headaches. Curr. Pain Headache Rep. 6, 320-323. https://doi.org/10.1007/s11916-002-0054-1
  21. Mauskop, A. and Basdeo, R. (2000) Botulinum toxin A is an effective prophylactic therapy for migraines. Cephalagia 20, 422.
  22. Montecucco, C. and Schiavo, G. (1994) Mechanism of action of tetanus and botulinum neurotoxins. Mol. Microbiol. 13, 1-8. https://doi.org/10.1111/j.1365-2958.1994.tb00396.x
  23. Morris, M. C., Depollier, J., Mery, J., Heitz, F. and Divita, G. (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19, 1173-1176. https://doi.org/10.1038/nbt1201-1173
  24. Oguma, K., Fujinaga, Y. and Inoue, K. (1995) Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39, 161-168. https://doi.org/10.1111/j.1348-0421.1995.tb02184.x
  25. Prochiantz, A. (2000) Messenger proteins: homeoproteins, TAT and others. Curr. Opin. Cell Biol. 12, 400-406. https://doi.org/10.1016/S0955-0674(00)00108-3
  26. Sarrabayrouse, M. A. M. (2002) Indications and limitations for the use of botulinum toxin for the treatment of facial wrinkles. Aesth. Plast. Surg. 26, 233-238. https://doi.org/10.1007/s00266-002-2030-x
  27. Schiavo, G., Matteoli, M. and Montecucco, C. (2000) Neurotoxins affecting neuroexocytosis. Physiol. Rev. 80, 717-766. https://doi.org/10.1152/physrev.2000.80.2.717
  28. Schmidt, J. J. and Bostian, K. A. (1995) Proteolysis of synthetic peptides by type A botulinum neurotoxin. J. Protein Chem. 14, 703-708. https://doi.org/10.1007/BF01886909
  29. Sugiyama, H. (1980) Clostidium botulinum neurotoxin. Microbiol. Rev. 44, 419-448.
  30. Verheyden, J. and Blitzer, A. (2002) Other noncosmetic uses of BOTOX. Dis. Mon. 48, 357-366. https://doi.org/10.1053/mda.2001.25136
  31. Vives, E., Brodin, P. and Lebleu, B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010-16017. https://doi.org/10.1074/jbc.272.25.16010
  32. Wadia, J. S. and Dowdy, S. F. (2002) Protein transduction technology. Curr. Opin. Biotechnol. 13, 52-56. https://doi.org/10.1016/S0958-1669(02)00284-7
  33. Wheeler, A. H. (1997) Therapeutic uses of botulinum toxin. Am. Fam. Physican 55, 541-545, 548.

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