Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Production of a Fusion Protein Containing the Antigenic Domain 1 of Human Cytomegalovirus Glycoprotein B

  • Sousa Fani (Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior) ;
  • Ferreira Susana (Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior) ;
  • Queiroz Joao (Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior) ;
  • Domingues Fernanda (Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior)
  • Published : 2006.07.01
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Abstract

The optimization of the production of a fusion protein containing the antigenic domain 1 (AD-1) is of a great importance, considering its use in diagnostic tests. The fusion protein is produced by the fermentation of a recombinant strain of Escherichia coli containing the plasmid Mbg58, which expresses the AD-1 (aa 484-650) of human cytomegalovirus glycoprotein B as a fusion protein together with aa 1-375 of ${\beta}-galactosidase$. An important characteristic of promoters (lac and derivatives) used in recombinant protein production in E. coli is their inducibility. Induction by IPTG is widely used for basic research; however, its use in large-scale production is undesirable because of its high cost and toxicity. In this work, studies using different inducers and carbon sources for the production of a fusion protein containing the AD-l were performed. The results showed that lactose could be used as an inducer in the fermentation process for the production of this protein, and that expression levels could exceed those achieved with IPTG. The use of lactose for protein expression in E. coli should be extremely useful for the inexpensive, large-scale production of heterologous proteins in E. coli. Addition of sucrose to the fermentation medium improved the yield of recombinant protein, whereas addition of fructose or trehalose decreased the yield.

Keywords

References

  1. Baneyx, F. 1999. Recombinant protein expression In Escherichia coli. Curr. Opin. Biotechnol. 10: 411-421 https://doi.org/10.1016/S0958-1669(99)00003-8
  2. Bhandari, P. and J. Gowrishankar. 1997. An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCI as the inducer. J. Bacteriol. 179: 44034406
  3. Britt, W. and M. Mach. 1996. Human Cytomegalovirus glycoproteins. Intervirology 39: 401-412 https://doi.org/10.1159/000150510
  4. Butler, S. and J. Falke. 1996. Effects of protein stabilizing agents on thermal backbone motions: A disulfide trapping study. Biochemistry 35: 10595-10600 https://doi.org/10.1021/bi961107v
  5. Donovan, R., C. Robinson, and B. R. Glick. 1996. Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. J. Ind. Microbiol. 16: 145-154 https://doi.org/10.1007/BF01569997
  6. Elbein, A. D., Y. T. Pan, I. Pastuszak, and D. Carrol. 2003. New insights on trehalose: A multifuntional molecule. Glycobiology 13: 17R-27R https://doi.org/10.1093/glycob/cwg047
  7. Ferreira, S., F. Sousa, J. A. Queiroz, and F. C. Domingues. 2005. Improved recovery of a fusion protein containing the antigenic domain 1 of the human cytomegalovirus glycoprotein B. Biotechnol. Lett. 27: 1241-1245 https://doi.org/10.1007/s10529-005-0024-x
  8. Gombert, A. K. and B. V. Kilikian. 1998. Recombinant gene expression in Escherichia coli cultivation using lactose as inducer. J. Biotechnol. 60: 47-54 https://doi.org/10.1016/S0168-1656(97)00185-5
  9. Khlebnicov, A. and J. Keasling. 2002. Effect of lac Y expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers. Biotech. Prog. 18: 672-674 https://doi.org/10.1021/bp010141k
  10. Kilikian, B. V., I. D. Suárez, C. W. Liria, and A. K. Gombert. 2000. Process strategies to improve heterologous protein production in Escherichia coli under lactose or IPTG induction. Process Biochem. 35: 1019-1025 https://doi.org/10.1016/S0032-9592(00)00137-0
  11. Kropff, B., M. Landini, and M. Mach. 1993. An ELISA using recombinant proteins for the detection of neutralizing antibodies against human cytomegalovirus. J. Med. Virol. 39: 187-195 https://doi.org/10.1002/jmv.1890390303
  12. Luli, G. W. and W. R. Strohl. 1990. Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl. Environ. Microbiol. 56: 1004-1011
  13. Menzella, H. G., E. A. Ceccarelli, and H. C. Gramajo. 2003. Novel Escherichia coli strain allows efficient recombinant protein production using lactose as inducer. Biotechnol. Bioeng. 82: 809-817 https://doi.org/10.1002/bit.10630
  14. Miao, F. and D. S. Kompala. 1992. Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter. I. Batch cultures and kinetic modelling. Biotechnol. Bioeng. 40: 787-796 https://doi.org/10.1002/bit.260400706
  15. Ohlin, M., V. Sundqvist, M. Mach, B. Wahren, and C. Borrebaeck. 1993. Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies. J. Virol. 67: 703-710
  16. Speckner, A., D. Glykofrydes, M. Ohlin, and M. Mach. 1999. Antigenic domain 1 of the human cytomegalovirus glycoprotein B induces a multitude of different antibodies which, when combined, results in incomplete virus neutralization. J. Gen. Virol. 80: 2183-2191 https://doi.org/10.1099/0022-1317-80-8-2183
  17. Speckner, A., B. Kropff, S. Knor, and M. Mach. 2000. The antigenic domain 1 of the cytomegalovirus glycoprotein B contains an intramolecular disulphide bond. J. Gen. Virol. 81: 2659-2663 https://doi.org/10.1099/0022-1317-81-11-2659
  18. Viitanen, M., A. Vasala, P. Nevbaner, and T. Alatossava. 2003. Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli. Microb. Cell Fact. 2: 1-10 https://doi.org/10.1186/1475-2859-2-1
  19. Woyski, D. and J. R. Cupp-Vickery. 2001. Enhanced expression of cytochrome P450s from lac-based plasmids using lactose as the inducer. Arch. Biochem. Biophys. 388: 276-280 https://doi.org/10.1006/abbi.2001.2306