DOI QR코드

DOI QR Code

Development of a Highly Efficient Protein-Secreting System in Recombinant Lactobacillus casei

  • Kajikawa, Akinobu (Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University) ;
  • Ichikawa, Eiko (Division of Biomedical Food Research, National Institute of Health Sciences) ;
  • Igimi, Shizunobu (Division of Biomedical Food Research, National Institute of Health Sciences)
  • Published : 2010.02.28

Abstract

The available techniques for heterologous protein secretion in Lactobacillus strains are limited. The aim of the present study was to develop an efficient protein-secretion system using recombinant lactobacilli for various applications such as live delivery of biotherapeutics. For the construction of expression vectors, the Lactobacillus brevis slpA promoter, Lactobacillus casei prtP signal sequence, and mouse IL-10 sequences were used as a model system. Interestingly, the slpA promoter exhibited strong activity in L. casei, contrary to previous observations. In order to stabilize replication of the plasmid in E. coli, a removable terminator sequence was built into the promoter region. For the improvement of secretion efficiency, a DTNSD oligopeptide was added to the cleavage site of signal peptidase. The resulting plasmids provided remarkably efficient IL-10 secretion. Accumulation of the protein in the culture supernatant varied widely according to the pH conditions. By analysis of the secreted protein, formation of homodimers, and biological activity, IL-10 was confirmed to be functional. The presently constructed plasmids could be useful tools for heterologous protein secretion in L. casei.

Keywords

References

  1. Acedo-Felix, E. and G. Perez-Martínez. 2003. Significant differences between Lactobacillus casei subsp. casei ATCC $393^T$ and a commonly used plasmid-cured derivative revealed by a polyphasic study. Int. J. Syst. Evol. Microbiol. 53: 67-75. https://doi.org/10.1099/ijs.0.02325-0
  2. Bernard, N., T. Ferain, D. Garmyn, P. Hols, and J. Delcour. 1991. Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli. FEBS Lett. 290: 61-64. https://doi.org/10.1016/0014-5793(91)81226-X
  3. Braat, H., P. Rottiers, D. W. Hommes, N. Huyghebaert, E. Remaut, J. P. Remon, et al. 2006. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin. Gastroenterol. Hepatol. 4: 754-759. https://doi.org/10.1016/j.cgh.2006.03.028
  4. Chang, T. L., C. H. Chang, D. A. Simpson, Q. Xu, P. K. Martin, L. A. Lagenaur, et al. 2003. Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc. Natl. Acad. Sci. U.S.A. 100: 11672-11677. https://doi.org/10.1073/pnas.1934747100
  5. Hazebrouck, S., R. Oozeer, K. Adel-Patient, P. Langella, S. Rabot, J. M. Wal, and G. Corthier. 2006. Constitutive delivery of bovine beta-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei. Appl. Environ. Microbiol. 72: 7460-7467. https://doi.org/10.1128/AEM.01032-06
  6. Kahala, M., K. Savijoki, and A. Palva. 1997. In vivo expression of the Lactobacillus brevis S-layer gene. J. Bacteriol. 179: 284-286.
  7. Kajikawa, A., E. Satoh, R. J. Leer, S. Yamamoto, and S. Igimi. 2007. Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis. Vaccine 25: 3599-3605. https://doi.org/10.1016/j.vaccine.2007.01.055
  8. Kruger, C., Y. Hu, Q. Pan, H. Marcotte, A. Hultberg, D. Delwar, et al. 2002. In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat. Biotechnol. 20: 702-706. https://doi.org/10.1038/nbt0702-702
  9. Le Loir, Y., A. Gruss, S. D. Ehrlich, and P. Langella. 1998. A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. J. Bacteriol. 180: 1895-1903.
  10. Le Loir, Y., S. Nouaille, J. Commissaire, L. Bretigny, A. Gruss, and P. Langella. 2001. Signal peptide and propeptide optimization for heterologous protein secretion in Lactococcus lactis. Appl. Environ. Microbiol. 67: 4119-4127. https://doi.org/10.1128/AEM.67.9.4119-4127.2001
  11. Maassen, C. B., J. D. Laman, C. van Holten-Neelen, L. Hoogteijling, L. Groenewegen, L. Visser, M. M. Schellekens, W. J. Boersma, and E. Claassen. 2003. Reduced experimental autoimmune encephalomyelitis after intranasal and oral administration of recombinant lactobacilli expressing myelin antigens. Vaccine 21: 4685-4693. https://doi.org/10.1016/S0264-410X(03)00522-X
  12. Maassen, C. B., C. van Holten-Neelen, F. Balk, M. J. den Bak-Glashouwer, R. J. Leer, J. D. Laman, W. J. Boersma, and E. Claassen. 2000. Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains. Vaccine 18: 2613-2623. https://doi.org/10.1016/S0264-410X(99)00378-3
  13. Mohamadzadeh, M., S. Olson, W. V. Kalina, G. Ruthel, G. L. Demmin, K. L. Warfield, S. Bavari, and T. R. Klaenhammer. 2005. Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc. Natl. Acad. Sci. U.S.A. 102: 2880-2885. https://doi.org/10.1073/pnas.0500098102
  14. Naes, H., J. A. Chrzanowsk, and H. Blom. 1991. Partial purification and characterization of a cell wall bound proteinase from Lactobacillus casei. Food Chem. 42: 65-79. https://doi.org/10.1016/0308-8146(91)90007-B
  15. Posno, M., P. T. Heuvelmans, M. J. van Giezen, B. C. Lokman, R. J. Leer, and P. H. Pouwels. 1991. Complementation of the inability of Lactobacillus strains to utilize D-xylose with Dxylose catabolism-encoding genes of Lactobacillus pentosus. Appl. Environ. Microbiol. 57: 2764-2766.
  16. Pouwels, P. H., A. Vriesema, B. Martinez, F. J. Tielen, J. F. Seegers, R. J. Leer, J. Jore, and E. Smit. 2001. Lactobacilli as vehicles for targeting antigens to mucosal tissues by surface exposition of foreign antigens. Methods Enzymol. 336: 369-389. https://doi.org/10.1016/S0076-6879(01)36602-8
  17. Pusch, O., D. Boden, S. Hannify, F. Lee, L. D. Tucker, M. R. Boyd, J. M. Wells, and B. Ramratnam. 2005. Bioengineering lactic acid bacteria to secrete the HIV-1 virucide cyanovirin. J. Acquir. Immune Defic. Syndr. 40: 512-220. https://doi.org/10.1097/01.qai.0000187446.76579.d3
  18. Savijoki, K., M. Kahala, and A. Palva. 1997. High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals. Gene 186: 255-262. https://doi.org/10.1016/S0378-1119(96)00717-2
  19. Schotte, L., L. Steidler, J. Vandekerckhove, and E. Remaut. 2000. Secretion of biologically active murine interleukin-10 by Lactococcus lactis. Enzyme Microb. Technol. 27: 761-765. https://doi.org/10.1016/S0141-0229(00)00297-0
  20. Shin, J. Y., W. M. Jeon, G. B. Kim, and B. H. Lee1. 2004. Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG. J. Dairy Sci. 87: 4097-4103. https://doi.org/10.3168/jds.S0022-0302(04)73552-3
  21. Steidler, L., W. Hans, L. Schotte, S. Neirynck, F. Obermeier, W. Falk, W. Fiers, and E. Remaut. 2000. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289: 1352-1355. https://doi.org/10.1126/science.289.5483.1352
  22. Syto, R., N. J. Murgolo, E. H. Braswell, P. Mui, E. Huang, and W. T. Windsor. 1998. Structural and biological stability of the human interleukin-10 homodimer. Biochemistry 37: 16943-16951. https://doi.org/10.1021/bi981555y
  23. Taguchi, H. and T. Ohta. 1991. D-Lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum. J. Biol. Chem. 266: 12588-12594.
  24. Tan, J. C., S. R. Indelicato, S. K. Narula, P. J. Zavodny, and C. C. Chou. 1993. Characterization of interleukin-10 receptors on human and mouse cells. J. Biol. Chem. 268: 21053-21059.
  25. Thompson-Snipes, L., V. Dhar, M. W. Bond, T. R. Mosmann, K. W. Moore, and D. M. Rennick. 1991. Interleukin 10: A novel stimulatory factor for mast cells and their progenitors. J. Exp. Med. 173: 507-510. https://doi.org/10.1084/jem.173.2.507
  26. Vidgren, G., I. Palva, R. Pakkanen, K. Lounatmaa, and A. Palva. 1992. S-Layer protein gene of Lactobacillus brevis: Cloning by polymerase chain reaction and determination of the nucleotide sequence. J. Bacteriol. 174: 7419-7427.
  27. Zdanov, A. 2004. Structural features of the interleukin-10 family of cytokines. Curr. Pharm. Des. 10: 3873-3884. https://doi.org/10.2174/1381612043382602

Cited by

  1. Optimisation of signal peptide for recombinant protein secretion in bacterial hosts vol.97, pp.9, 2010, https://doi.org/10.1007/s00253-013-4831-z
  2. Characterization of flagellins isolated from a highly motile strain of Lactobacillus agilis vol.16, pp.None, 2010, https://doi.org/10.1186/s12866-016-0667-x
  3. Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications vol.15, pp.None, 2010, https://doi.org/10.1186/s12934-016-0468-9
  4. Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications vol.9, pp.None, 2018, https://doi.org/10.3389/fmicb.2018.02264
  5. Safety and efficacy of mucosal immunotherapy using human papillomavirus (HPV) type 16 E7-expressing Lactobacillus -based vaccine for the treatment of high-grade squamous intraepithelial lesion (HSIL) vol.49, pp.9, 2010, https://doi.org/10.1093/jjco/hyz095
  6. Natural and engineered promoters for gene expression in Lactobacillus species vol.104, pp.9, 2020, https://doi.org/10.1007/s00253-020-10426-0