1887

Abstract

One of the limitations facing the development of into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

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2009-11-01
2019-12-16
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References

  1. Ayakawa, G. Y., Boushell, L. W., Crowley, L. W., Erdos, P. J., Mc, G. W., Arthur, W. P. & Bleiweis, A. S. ( 1987; ). Isolation and characterization of monoclonal antibodies specific for antigen P1, a major surface protein of mutans streptococci. Infect Immun 55, 2759–2767.
    [Google Scholar]
  2. Baca, A. M. & Hol, W. G. ( 2000; ). Overcoming codon bias: a method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli. Int J Parasitol 30, 113–118.[CrossRef]
    [Google Scholar]
  3. Brinkmann, U., Mattes, R. E. & Buckel, P. ( 1989; ). High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene 85, 109–114.[CrossRef]
    [Google Scholar]
  4. Gustafsson, C., Govindarajan, S. & Minshull, J. ( 2004; ). Codon bias and heterologous protein expression. Trends Biotechnol 22, 346–353.[CrossRef]
    [Google Scholar]
  5. Halperin, S. A., Issekutz, T. B. & Kasina, A. ( 1991; ). Modulation of Bordetella pertussis infection with monoclonal antibodies to pertussis toxin. J Infect Dis 163, 355–361.[CrossRef]
    [Google Scholar]
  6. High, K. P., Joiner, K. A. & Handschumacher, R. E. ( 1994; ). Isolation, cDNA sequences, and biochemical characterization of the major cyclosporine-binding proteins of Toxoplasma gondii. J Biol Chem 269, 9105–9112.
    [Google Scholar]
  7. Homonylo-McGavin, M. K. & Lee, S. F. ( 1996; ). Role of the C terminus in antigen P1 surface localization in Streptococcus mutans and two related cocci. J Bacteriol 178, 801–807.
    [Google Scholar]
  8. Hua, Z., Wang, H., Chen, D., Chen, Y. & Zhu, D. ( 1994; ). Enhancement of expression of human granulocyte-macrophage colony stimulating factor by argU gene product in Escherichia coli. Biochem Mol Biol Int 32, 537–543.
    [Google Scholar]
  9. Kane, J. F. ( 1995; ). Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6, 494–500.[CrossRef]
    [Google Scholar]
  10. Kim, S. & Lee, S. B. ( 2006; ). Rare codon clusters at 5′-end influence heterologous expression of archaeal gene in Escherichia coli. Protein Expr Purif 50, 49–57.[CrossRef]
    [Google Scholar]
  11. Knight, J. B., Halperin, S. A., West, K. A. & Lee, S. F. ( 2008; ). Expression of a functional single chain variable fragment antibody against the complement receptor 1 in Streptococcus gordonii. Clin Vaccine Immunol 15, 925–931.[CrossRef]
    [Google Scholar]
  12. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  13. LeBlanc, D. J., Lee, L. N. & Abu-Al-Jaibat, A. ( 1992; ). Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin. Plasmid 28, 130–145.[CrossRef]
    [Google Scholar]
  14. Lee, S. F. ( 2003; ). Oral colonization and immune responses to Streptococcus gordonii: potential use as a vector to induce antibody against respiratory pathogens. Curr Opin Infect Dis 16, 231–235.[CrossRef]
    [Google Scholar]
  15. Lee, S. F., Progulske-Fox, A., Erdos, G. W., Ayakawa, G. A., Crowley, P. J. & Bleiweis, A. S. ( 1989; ). Construction and characterization of isogenic mutants of Streptococcus mutans deficient in the major surface protein antigen P1. Infect Immun 57, 3306–3313.
    [Google Scholar]
  16. Lee, S. F., March, R., Halperin, S. A., Faulkner, G. & Gao, L. Q. ( 1999; ). Surface expression of a protective recombinant pertussis toxin S1 subunit fragment in Streptococcus gordonii. Infect Immun 67, 1511–1516.
    [Google Scholar]
  17. Lee, S. F., Halperin, S. A., Wang, H. & MacArthur, A. ( 2002; ). Oral colonization and immune responses to Streptococcus gordonii expressing a pertussis toxin S1 fragment in mice. FEMS Microbiol Lett 208, 175–178.[CrossRef]
    [Google Scholar]
  18. Li, A., Kato, Z., Ohnishi, H., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y. & Kondo, N. ( 2003; ). Optimized gene synthesis and high expression of human interleukin-18. Protein Expr Purif 32, 110–118.[CrossRef]
    [Google Scholar]
  19. Mallaley, P. P., Halperin, S. A., Morris, A., MacMillian, A. & Lee, S. F. ( 2006; ). Expression of pertussis toxin S1 fragment by inducible promoters in oral streptococci and immune responses elicited during oral colonization in mice. Can J Microbiol 52, 436–444.[CrossRef]
    [Google Scholar]
  20. Medaglini, D., Rush, C. M., Sestini, P. & Pozzi, G. ( 1997; ). Commensal bacteria as vectors for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci induces local and systemic antibodies in mice. Vaccine 15, 1330–1337.[CrossRef]
    [Google Scholar]
  21. Peterson, S., Cline, R. T., Tettelin, H., Sharov, V. & Morrison, D. A. ( 2000; ). Gene expression analysis of the Streptococcus pneumoniae competence regulons by use of DNA microarrays. J Bacteriol 182, 6192–6202.[CrossRef]
    [Google Scholar]
  22. Sorensen, M., Lippuner, C., Kaiser, T., Misslitz, A., Aebischer, T. & Bumann, D. ( 2003; ). Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria. FEBS Lett 552, 110–114.[CrossRef]
    [Google Scholar]
  23. Tjalsma, H., Antelmann, H., Jongbloed, J. D., Braun, P. G., Darmon, E., Dorenbos, R., Dubois, J. Y., Westers, H., Zanen, G. & other authors ( 2004; ). Proteomics of protein secretion by Bacillus subtilis: separating the “secrets” of the secretome. Microbiol Mol Biol Rev 68, 207–233.[CrossRef]
    [Google Scholar]
  24. Towbin, H., Staehelin, T. & Gordon, J. ( 1979; ). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.[CrossRef]
    [Google Scholar]
  25. Zdanovsky, A. G. & Zdanovkaia, M. V. ( 2000; ). Simple and efficient method for heterologous expression of clostridial proteins. Appl Environ Microbiol 66, 3166–3173.[CrossRef]
    [Google Scholar]
  26. Zhou, Z., Schnake, P., Xiao, L. & Lal, A. A. ( 2004; ). Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization. Protein Expr Purif 34, 87–94.[CrossRef]
    [Google Scholar]
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