Journal of Microbiology and Biotechnology

  • 제19권4호
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  • Pages.416-423
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  • 2009
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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A Preparative Purification Process for Recombinant Hepatitis B Core Antigen Using Online Capture by Expanded Bed Adsorption Followed by Size-Exclusion Chromatography

  • Ho, Chin Woi (Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia) ;
  • Tan, Wen Siang (Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia) ;
  • Chong, Fui Chin (Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia) ;
  • Ling, Tau Chuan (Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia) ;
  • Tey, Beng Ti (Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia)
  • 발행 : 2009.04.30
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초록

Hepatitis B core antigen(HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus(HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.

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참고문헌

  1. Betancourt, A. A., C. A. G. Delgado, Z. C. Estevez, J. C. Martínez, G. Veliz Ríos, S. R. M. Aureoles-Rosello, et al. 2007. Phase I clinical trial in healthy adults of a nasal vaccine candidate containing recombinant hepatitis B surface and core antigens. Int. J. Infect. Dis. 11: 394-401 https://doi.org/10.1016/j.ijid.2006.09.010
  2. Bierau, H., R. J. Hinton, and A. Lyddiatt. 2001. Direct process integration of cell disruption and fluidised bed adsorption in the recovery of labile microbial enzymes. Bioseparation 10: 73-85 https://doi.org/10.1023/A:1012054622066
  3. Bierau, H., Z. Zhang, and A. Lyddiatt. 1999. Direct process integration of cell disruption and fluidised bed adsorption for the recovery of intracellular proteins. J. Chem. Technol. Biotech. 74: 208-212 https://doi.org/10.1002/(SICI)1097-4660(199903)74:3<208::AID-JCTB21>3.0.CO;2-P
  4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  5. Broos, K., P. Vanlandschoot, M. Maras, J. Robbens, G. Leroux-Roels, and Y. Guisez. 2007. Expression, purification and characterization of full-length RNA-free hepatitis B core particles. Protein Exp. Purif. 54: 30-37 https://doi.org/10.1016/j.pep.2007.02.006
  6. Bottcher, B., S. A. Wynne, and R. A. Crowther. 1997. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 386: 88-91 https://doi.org/10.1038/386088a0
  7. Cabanne, C., A. M. Noubhani, W. Dieryck, A. Hocquellet, and X. Santarelli. 2004. Evaluation of three expanded bed adsorption anion exchange matrices with the aid of recombinant enhanced green fluorescent protein overexpressed in Escherichia coli. J. Chromatogr. B 808: 91-97 https://doi.org/10.1016/j.jchromb.2004.02.009
  8. Chen, H., J. H. Lu, W. Q. Liang, Y. H. Huang, W. J. Zhang, and D. B. Zhang. 1994. Purification of the recombinant hepatitis B virus core antigen (rHBcAg) produced in the yeast Saccharomyces cerevisiae and comparative observation of its particles by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Micron 35: 311-318 https://doi.org/10.1016/j.micron.2003.12.003
  9. Cohen, B. J. and J. E. Richmond. 1982. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature 296: 277-278 https://doi.org/10.1038/296277a0
  10. Cooper, A. and Y. Shaul. 2005. Recombinant viral capsids as an efficient vehicle of oligonucleotide delivery into cells. Biochem. Biophys. Res. Commun. 327: 1094-1099 https://doi.org/10.1016/j.bbrc.2004.12.118
  11. Crowther, R. A., N. A. Kiselev, B. Bottcher, J. A. Berriman, G. P. Borisova, V. Ose, and P. Pumpens. 1994. Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell 77: 943-950 https://doi.org/10.1016/0092-8674(94)90142-2
  12. Dyson, M. R. and K. Murray. 1995. Selection of peptide inhibitors of interactions involved in complex protein assemblies: Association of the core and surface antigens of hepatitis B virus. Proc. Natl. Acad. Sci. USA 92: 2194-2198 https://doi.org/10.1073/pnas.92.6.2194
  13. Fiers, W., M. D. Filette, A. Birkett, S. Neirynck, and W. M. Jou. 2004. A "universal" human influenza A vaccine. Virus Res. 103: 173-176 https://doi.org/10.1016/j.virusres.2004.02.030
  14. Hackstadt, T., W. J. Todd, and H. D. Caldwell. 1985. Disulfidemediated nteractions of the chlamydial major outer membrane protein: Role in the differentiation of chlamydiae? J. Bacteriol. 161: 25-31
  15. Hamilton, G. E., F. Luechau, S. C. Burton, and A. Lyddiatt. 2000. Development of a mixed mode adsorption process for the direct product sequestration of an extracellular protease from microbial batch cultures. J. Biotechnol. 79: 103-115 https://doi.org/10.1016/S0168-1656(99)00242-4
  16. Ho, C. W., T. K. Chew, T. C. Ling, S. Kamaruddin, W. S. Tan, and B. T. Tey. 2006. Efficient mechanical cell disruption of Escherichia coli by an ultrasonicator and recovery of intracellular hepatitis B core antigen. Process Biochem. 41: 1829-1834 https://doi.org/10.1016/j.procbio.2006.03.043
  17. Ho, C. W., W. S. Tan, S. Kamarudin, T. C. Ling, and B. T. Tey. 2007. The release of hepatitis B core antigen from Escherichia coli by batch mode bead milling. Process Biochem. 43: 206- 212 https://doi.org/10.1016/j.procbio.2007.11.004
  18. Imamura, T., K. Sugahara, S. Adachi, Y. Miyatsu, H. Mizokami, and T. Matsusaka. 1988. Purification and characterization of the hepatitis B virus core antigen produced in the yeast Saccharomyces cerevisiae. J. Biotechnol. 8: 149-161 https://doi.org/10.1016/0168-1656(88)90077-6
  19. Kaufmann, M. 1997. Unstable proteins: How to subject them to chromatographic separations for purification procedures. J. Chromatogr. B 699: 347-369 https://doi.org/10.1016/S0378-4347(96)00512-9
  20. Kim, K., H.-J. Kim, D.-K. Oh, J.-J. Cheong, and S. Rhee. 2006. Expression, purification, and crystallization of D-psicose 3- epimerase from Agrobacterium tumefaciens. J. Microbiol. Biotechnol. 16: 647-650
  21. Kim, S.-W, H.-J. Hwang, J.-W. Choi, and J.-W. Yun. 2005. Characterization of exopolysaccharides produced by submerged culture of an entomopathogenic fungus Paecilomyces sinclairii by using a multi-angle laser light scattering system. J. Microbiol. Biotechnol. 15: 560-567
  22. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680- 685 https://doi.org/10.1038/227680a0
  23. Ling, T. C. and A. Lyddiatt. 2005. Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: A comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems. J. Biotechnol. 119: 436-448 https://doi.org/10.1016/j.jbiotec.2005.05.029
  24. Loa, C. C., T. L. Lin, C. C. Wu, T. A. Bryan, H. L. Thacker, T. Hooper, and D. Schrader. 2002. Purification of turkey coronavirus by Sephacryl size-exclusion chromatography. J. Virol. Methods 104: 187-194 https://doi.org/10.1016/S0166-0934(02)00069-1
  25. Morton, P. and A. Lyddiatt. 1994. Direct integration of protein recovery with productive fermentations, pp. 329-335. In D. L. Pyle (ed.), Separations for Biotechnology 3. Royal Society of Chemistry, Cambridge, U.K
  26. Nassal, M., C. Skamel, M. Vogel, P. A. Kratz, T. Stehle, R. Wallich, and M. M. Simon. 2008. Development of hepatitis B virus capsids into a whole-chain protein antigen display platform: New particulate Lyme disease vaccines. Int. J. Med. Microbiol. 298: 135-142 https://doi.org/10.1016/j.ijmm.2007.08.002
  27. Ng, M. Y. T., W. S. Tan, N. Abdullah, T. C. Ling, and B T. Tey. 2006. Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen. J. Virol. Methods 137: 134-139 https://doi.org/10.1016/j.jviromet.2006.06.016
  28. Park, S., H. Cho, Y. Kim, S. Ahn, and T. Chang. 2007. Fast size-exclusion chromatography at high temperature. J. Chromatogr. A 1157: 96-100 https://doi.org/10.1016/j.chroma.2007.04.049
  29. Pumpens, P. and E. Grens. 1999. Hepatitis B core particles as a universal display model: A structure-function basis for development. FEBS Lett. 442: 1-6 https://doi.org/10.1016/S0014-5793(98)01599-3
  30. Rojas, E. E. G., J. S. D. R. Coimbra, L. A. Minim, A. D. G. Zuniga, S. H. Saraiva, and V. P. R. Minim. 2004. Size-exclusion chromatography applied to the purification of whey proteins from the polymeric and saline phases of aqueous two-phase systems. Process Biochem. 39: 1751-1759 https://doi.org/10.1016/j.procbio.2003.07.002
  31. Rolland, D., M. Gauthier, C. Dugua, C. Fournier, L. Delpech, B. Watelet, O. Letourneur, M. Arnaud, and M. Jolivet. 2001. Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: Comparison of size exclusion chromatography and ultracentrifugation. J. Chromatogr. B 753: 51-65 https://doi.org/10.1016/S0378-4347(00)00538-7
  32. Sahin, A., E. Tetaud, G. Merlin, and X. Santarelli. 2005. LdARL- 1 His-tagged recombinant protein: Purification by immobilized metal affinity expanded bed adsorption. J. Chromatogr. B 818: 19-22 https://doi.org/10.1016/j.jchromb.2004.10.022
  33. Sun, G., S. Pal, A. K. Sarcon, S. Kim, E. Sugawara, H. Nikaido, M. J. Cocco, E. M. Peterson, and L. M. de la Maza. 2007. Structural and functional analyses of the major outer membrane protein of Chlamydia trachomatis. J. Bacteriol. 189: 6222- 6235 https://doi.org/10.1128/JB.00552-07
  34. Tan, W. S., I. W. McNae, K. L. Ho, and M. D. Walkinshaw. 2007. Crystallization and X-ray analysis of the T=4 particle of hepatitis B capsid protein with an N-terminal extension. Acta Cryst. F 63: 642-647 https://doi.org/10.1107/S1744309107033726
  35. Tan, W. S., M. R. Dyson, and K. Murray. 1999. Two distinct segments of the hepatitis B virus surface antigen contribute synergistically to its association with the viral core particles. J. Mol. Biol. 286: 797-808 https://doi.org/10.1006/jmbi.1998.2525
  36. Tan, W. S., M. R. Dyson, and K. Murray. 2003. Hepatitis B virus core antigen: Enhancement of its production in Escherichia coli, and interaction of the core particles with the viral surface antigen. Biol. Chem. 384: 363-371 https://doi.org/10.1515/BC.2003.042
  37. Tang, K. F., M. P. Abdullah, K. Yusoff, and W. S. Tan. 2007. Interactions of hepatitis B core antigen and peptide inhibitors. J. Med. Chem. 50: 5620-5626 https://doi.org/10.1021/jm070468d
  38. Tey, B. T., K. H. Yong, H. P. Ong, T. C. Ling, S. T. Ong, Y. P. Tan, A. Ariff, and W. S. Tan. 2004. Optimal conditions for hepatitis B core antigen production in shaked flask fermentation. Biotechnol. Bioprocess Eng. 9: 374-378 https://doi.org/10.1007/BF02933060
  39. Thwaites, E., S. C. Burton, and A. Lyddiatt. 2002. Impact of the physical and topographical characteristics of adsorbent solidphases upon the fluidised bed recovery of plasmid DNA from Escherichia coli lysates. J. Chromatogr. A 943: 77-90 https://doi.org/10.1016/S0021-9673(01)01445-5
  40. Ulrich, R., M. Nassal, H. Meisel, and D. H. Kr$\ddot{u}$ger. 1998. Core particles of hepatitis B virus as carrier for foreign epitopes. Adv. Virus Res. 50: 141-182 https://doi.org/10.1016/S0065-3527(08)60808-8
  41. Wang, J., W. Chen, J. Lu, and S. Lu. 2003. Overexpression and purification of recombinant atrial natriuretic peptide using hybrid fusion protein REF-ANP in Escherichia coli. Protein Exp. Purif. 28: 49-56 https://doi.org/10.1016/S1046-5928(02)00611-3
  42. Wizemann, H. and A. V. Brunn. 1999. Purification of E. coliexpressed HIS-tagged hepatitis B core antigen by $Ni^{2+}$-chelate affinity chromatography. J. Virol. Methods 77: 189-197 https://doi.org/10.1016/S0166-0934(98)00152-9
  43. Zhang, Z., S. Burton, S. Williams, E. Thwaites, and A. Lyddiatt. 2001. Design and assembly of solid-phases for the effective recovery of nanoparticulate bioproducts in fluidised bed contactors. Bioseparation 10: 113-132 https://doi.org/10.1023/A:1012013404483
  44. Zheng, J., F. Sch$\ddot{o}$del, and D. L. Peterson. 1992. The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern. J. Biol. Chem. 267: 9422-9429

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