Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression of Functional Pentameric Heat-Labile Enterotoxin B Subunit of Escherichia coli in Saccharomyces cerevisiae

  • Lim, Jung-Gu (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Kim, Jung-Ae (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Chung, Hea-Jong (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Kim, Tae-Geum (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Kim, Jung-Mi (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Lee, Kyung-Ryul (Department of Microbiology and Institute of Oral Bioscience, Chonbuk National University) ;
  • Park, Seung-Moon (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Yang, Moon-Sik (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Kim, Dae-Hyuk (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University)
  • Published : 2009.05.31
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Abstract

Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmci reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked inmmunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active aligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.

Keywords

References

  1. Arakawa, T., D. K. Chong, J. L. Merritt, and W. H. Langridge. 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res. 6: 403-413 https://doi.org/10.1023/A:1018487401810
  2. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Brake, A. J., J. P. Merryweather, D. G. Coit, U. A. Heberlein, T. P. Masiary, G. T. Mullenback, M. S. Urdea, P. Valenzuela, and P. J. Barr. 1984. Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 81: 4642-4646 https://doi.org/10.1073/pnas.81.15.4642
  4. De Geus, B., M. Dol-Bosman, J. W. Scholten, W. Stok, and A. Bianchi. 1997. A comparison of natural and recombinant cholera toxin B subunit as stimulatory factors in intranasal immunization. Vaccine 15: 1110-1113 https://doi.org/10.1016/S0264-410X(97)00007-8
  5. Dickinson, B. L. and J. D. Clements. 1995. Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity. Infect. Immun. 63: 1617-1623
  6. Fattal-German, M. and B. Bizzini. 1992. Assessment of the anti-viral effect of a short-term oral treatment of mice with live Saccharomyces cerevisiae cells. Dev. Biol. Stand. 77: 115-120
  7. Feil, I. K., R. Reddy, L. de Haan, E. A. Merritt, F. van den Akker, D. R. Storm, and W. G. Hol. 1996. Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity. Mol. Microbiol. 20:823-832 https://doi.org/10.1111/j.1365-2958.1996.tb02520.x
  8. Fingerut, E., B. Gutter, R. Meir, D. Eliahoo, and J. Pitcovski. 2005. Vaccine and adjuvant activity of recombinant subunit B of E. coli enterotoxin produced in yeast. Vaccine 23: 4685-4696 https://doi.org/10.1016/j.vaccine.2005.03.050
  9. Ge, L. and P. Rudolph. 1997. Simultaneous introduction of multiple mutations using overlap extension PRC. Biotechniques 22: 28-30
  10. Goto, N., J. Maeyama, Y. Yasuda, M. Isaka, K. Matano, S. Kozuka, et al. 2000. Safety evaluation of recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant. Vaccine 18: 2164-2171 https://doi.org/10.1016/S0264-410X(99)00337-0
  11. Haan, L., W. Verweij, E. Agsteribbe, and J. Wilschut. 1998. The role of ADP-ribosylation and GM1-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin. Immunol. Cell Biol. 76: 270-279 https://doi.org/10.1046/j.1440-1711.1998.00745.x
  12. Hagiwar, Y., T. Tsuji, T. Iwasaki, S. Kadowaki, H. Asanuma, Z. Chen, et al. 2001. Effectiveness and safety of mutant Escherichia coli heat-labile enterotoxin(LT H44A) as an adjuvant for nasal influenza vaccine. Vaccine 19: 2071-2079 https://doi.org/10.1016/S0264-410X(00)00414-X
  13. Haq, T. A., H. S. Mason, J. D. Clements, and C. J. Arntzen. 1995. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268: 714-716 https://doi.org/10.1126/science.7732379
  14. Hayward, C. M., P. O'aora, D. B. Young, G. E. Griffin, J. Thole, T. R. Hirst, L. R. Castello-Branco, and D. J. Lewis. 1999. Construction and murine immunogenicity of recombinant Bacille Calmette Gu\'{e}rin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin. Vaccine 17: 1272-1281 https://doi.org/10.1016/S0264-410X(98)00350-8
  15. Hirst, T. R. 1991. Assembly and secretion of oligomeric toxins in Gram negative bacteria. In J. E. Alouf and J. H. Freer (eds.), A Source Book of Bacterial Protein Toxins. Academic Press, London
  16. Hirst, T. R. and J. Holmgren. 1987. Conformation of protein secreted across bacterial outer membranes: A study of enterotoxin translocation from Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 84: 7418-7422 https://doi.org/10.1073/pnas.84.21.7418
  17. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168
  18. Kang, T. J., S. C. Han, M. O. Jang, K. H. Kang, Y. S. Jang, and M. S. Yang. 2004. Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence. Appl. Biochem. Biotechnol. 117: 175-187 https://doi.org/10.1385/ABAB:117:3:175
  19. Kang, T. J., S. C. Han, M. S. Yang, and Y. S. Jang. 2006. Expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat-labile enterotoxin in tobacco plants. Protein Expr. Purif. 46: 16-22 https://doi.org/10.1016/j.pep.2005.07.026
  20. Katz, D. E., A. J. DeLorimier, and M. K. Wolf. 2003. Oral immunization of adult volunteers with microencapsulated enterotoxigenic E. coli (ETEC) CS6 antigen. Vaccine 21: 341-346 https://doi.org/10.1016/S0264-410X(02)00613-8
  21. Kim, T. G., M. Y. Kim, B. G. Kim, T. J. Kang, Y. S. Kim, Y. S. Jang, C. J. Arntzen, and M. S. Yang. 2007. Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa). Protein Expr. Purif. 51:22-27 https://doi.org/10.1016/j.pep.2006.05.024
  22. Liljeqvist, S., P. Samuelson, M. Hansson, T. N. Nguyen, H. Binz, and S. Stahl. 1997. Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus. Appl. Environ. Microbiol. 63: 2481-2488
  23. Lim, Y. Y., E. H. Park, J. H. Kim, S. M. Park, H. S. Jang, Y. J. Park, S. W. Yoon, M. S. Yang, and D. H. Kim. 2001. Enhanced and targeted expression of fungal phytase in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 11: 915-921
  24. Lim, Y. Y., M. Y. Lee, B. W. Chung, S. M. Park, S. G. Park, H. S. Jang, M. S. Yang, and D. H. Kim. 2002. Expression of a functional human interleukin-18 in yeast. Enz. Microbial Technol. 30: 703-709 https://doi.org/10.1016/S0141-0229(02)00043-1
  25. Lim, Y. Y., S. M. Park, Y. S. Jang, M. S. Yang, and D. H. Kim. 2003. Production of a functional mouse interferon from recombinant Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 13: 537-543
  26. Loison, G., A. Vidal, A. Findeli, C. Roitsch, J. M. Balloul, and Y. Lemoine. 1989. High level expression of a protective antigen of schistosomes in Saccharomyces cerevisiae. Yeast 5: 497-507 https://doi.org/10.1002/yea.320050609
  27. Lucke, N., T. Tsuji, and J. Holmgren. 1992. The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity. Eur. J. Immunol. 22: 2277-2281 https://doi.org/10.1002/eji.1830220915
  28. Merritt, E. A., S. E. Pronk, T. K. Sixma, K. H. Kalk, B. A. Van Zanten, and W. G. J. Hol. 1994. Structure of partially-activated E. coli heat-labile enterotoxin (LT) at 2.6 $\AA$ resolution. FEBS Lett. 337: 88-92 https://doi.org/10.1016/0014-5793(94)80635-7
  29. Mo, A. Y., Y. S. Kim, S. M. Park, M. S. Yang, and D. H. Kim. 2005. Expression of fungal phytase on the cell surface of Saccharomyces cerevisiae. Biotechnol. Bioprocess Eng. 10: 576-581 https://doi.org/10.1007/BF02932297
  30. Park, S. M., A. Y. Mo, Y. S. Jang, J. H. Lee, M. S. Yang, and D. H. Kim. 2004. Expression of a functional human tumor necrosis factor (hTNF)-$\alpha$ in yeast Saccharomyces cerevisiae. Biotech. Bioprocess Eng. 9: 292-296 https://doi.org/10.1007/BF02942346
  31. Pohl, T. 1990. Concentration of proteins and removal of solutes. Methods Enzymol. 182: 68-83 https://doi.org/10.1016/0076-6879(90)82009-Q
  32. Purvis, I. J., A. J. E. Bettany, L. Loughlin, and A. J. P. Brown. 1987. Translation and stability of an Escherichia coli betagalactosidase mRNA expressed under the control of pyruvate kinase sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 15: 7963-7974 https://doi.org/10.1093/nar/15.19.7963
  33. Rezaee, M. A., A. Rezaee, S. M. Moazzeni, A. H. Salmanian, Y. Yasuda, K. Tochikubo, S. N. Pirayeh, and M. Arzanlou. 2005. Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae. J. Microbiol. 43: 354-360
  34. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  35. Schellenberg, D., A. Bonington, C. M. Champion, R. Lancaster, S. Webb, and J. Main. 1994. Treatment of Clostridium difficile diarrhoea with brewer's yeast. Lancet 343: 171-172
  36. Schonberger, O., T. R. Hirst, and O. Pines. 1991. Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae. Mol. Microbiol. 5: 2663-2671 https://doi.org/10.1111/j.1365-2958.1991.tb01975.x
  37. Schreuder, M. P., C. Deen, W. J. A. Boersma, P. H. Pouwels, and F. M. Klis. 1996. Yeast expressing hepatitis B virus surface antigen determinants on its surface: Implications for a possible oral vaccine. Vaccine 14: 383-388 https://doi.org/10.1016/0264-410X(95)00206-G
  38. Shin, Y. M., T. H. Kwon, K. S. Kim, K. S. Chae, D. H. Kim, and M. S. Yang. 2001. Enhanced iron uptake of Saccharomyces cerevisiae by the heterologous expression of a tadpole ferritin gene. Appl. Env. Microbiol. 67: 1280-1283 https://doi.org/10.1128/AEM.67.3.1280-1283.2001
  39. Shon, J. H., E. S. Choi, B. H. Chung, D. J. Youn, and J. H. Seo. 1995. Process development of the production of recombinant hirudin in Saccharomyces cerevisiae: From upstream to downstream. Proc. Biochem. 30: 653-660 https://doi.org/10.1016/0032-9592(94)00062-X
  40. Sixma, T. K., A. Aguirre, A. C. Terwisscha van Scheltinga, E. S. Wartna, K. H. Kalk, and W. G. Hol. 1992. Heat-labile enterotoxin crystal forms with variable A/$B_5$ orientation: Analysis of conformational flexibility. FEBS Lett. 305: 81-85 https://doi.org/10.1016/0014-5793(92)80869-I
  41. Sixma, T. K., S. E. Pronk, K. H. Kalk, E. S. Wartna, B. A. van Zanten, B. Witholt, and W. G. Hol. 1991. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature 351: 371-377 https://doi.org/10.1038/351371a0
  42. Snider D. P. 1995. The mucosal adjuvant activities of ADPribosylating bacterial enterotoxins. Crit. Rev. Immunol. 15: 317-348 https://doi.org/10.1615/CritRevImmunol.v15.i3-4.70
  43. Spangler, B. D. 1992. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol. Rev. 56: 622-647
  44. Steinsland, H., P. Valentiner-Branth, H. K. Gjessing, P. Aaby, K. Molbak, and H. Sommerfelt. 2003. Protection from natural infections with enterotoxigenic Escherichia coli: Longitudinal study. Lancet 362: 286-291 https://doi.org/10.1016/S0140-6736(03)13971-2
  45. Tochikubo, K. and Y. Yasuda. 2000. Principle of mucosal immunity and development of mucosal vaccines using cholera toxin B subunit and its related adjuvants. Rec. Res. Devel. Microbiol. 4: 387-405
  46. Tsuji, T., K. Watanabe, and A. Miyama. 1995. Monomer of the B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli has little ability to bind to GM1 ganglioside compared to its coligenoid. Microbiol. Immunol. 39: 817-819 https://doi.org/10.1111/j.1348-0421.1995.tb03262.x
  47. Williams, N. A., T. R. Hirst, and T. O. Nashar. 1999. Immune modulation by the cholera-like enterotoxins: From adjuvant to therapeutic. Immunol. Today 20: 95-101 https://doi.org/10.1016/S0167-5699(98)01397-8
  48. Yamamoto, T., T. Tamura, M. Ryoji, A. Kaji, T. Yokota, and T. Takano. 1982. Sequence analysis of the heat-labile enterotoxin subunit B gene originating in human enterotoxigenic Escherichia coli. J. Bacteriol. 152: 506-509

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