A recombinant vaccine against hydatidosis: production of the antigen in Escherichia coli

  • Daniel Manderson
  • Robert Dempster
  • Yusuf ChistiEmail author
Original Paper


A commercial process was developed for producing a recombinant vaccine against hydatidosis in farm animals. The vaccine antigen consisting of a surface protein of the oncospheres of the hydatid worm (Echinococcus granulosus), was produced as inclusion bodies in Escherichia coli. Fed-batch cultures of E. coli using Terrific broth in stirred bioreactors at 37°C, pH 7.0, and a dissolved oxygen level of 30% of air saturation produced the highest volumetric concentrations of the final solubilized antigen. An exponential feeding strategy proved distinctly superior to feeding based on pH-stat and DO-stat methods. The plasmid coding for the antigen was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at 4 h after initiation of the culture. The minimum IPTG concentration for full induction was 0.1 mM.


Echinococcus granulosus Escherichia coli Hydatid Hydatidosis Recombinant vaccine Veterinary vaccine 


  1. Akesson M, Karlsson EN, Hagander P, Axelsson J, Tocaj A (1999) On-line detection of acetate formation in Escherichia coli cultures using dissolved oxygen responses to feed transients. Biotechnol Bioeng 64:590–598CrossRefPubMedGoogle Scholar
  2. Akesson M, Hagander P, Axelsson J (2001) Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding. Biotechnol Bioeng 73:223–230CrossRefPubMedGoogle Scholar
  3. Atlas RM (1997) Handbook of microbiological media, 2nd edn. CRC, New YorkGoogle Scholar
  4. Bhattacharya SK, Dubey AK (1997) Effect of dissolved oxygen and oxygen mass transfer on overexpression of target gene in recombinant E. coli. Enzyme Microb Technol 20:355–360CrossRefGoogle Scholar
  5. Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Castan A, Enfors S-O (2000) Characteristics of a DO-controlled fed-batch culture of Escherichia coli. Bioproc Eng 22:509–515CrossRefGoogle Scholar
  7. Chang CC, Ryu DDY, Park CS, Kim J-Y, Ogrydziak DM (1998) Recombinant bioprocess optimization for heterologous protein production using two-stage, cyclic fed-batch culture. Appl Microbiol Biotechnol 49:531–537CrossRefPubMedGoogle Scholar
  8. Chen Q, Bentley WE, Weigand WA (1995) Optimisation for a recombinant E. coli fed-batch fermentation. Appl Biochem Biotechnol 51:449–461PubMedCrossRefGoogle Scholar
  9. Chisti Y (1998) Strategies in downstream processing. In: Subramanian G (ed) Bioseparation and bioprocessing: a handbook, vol 2. Wiley-VCH, New York, pp 3–30Google Scholar
  10. Chisti Y (1999a) Solid substrate fermentation, enzyme production, food enrichment. In: Flickinger MC, Drew SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation. Wiley, New York, pp 2446–2462Google Scholar
  11. Chisti Y (1999b) Shear sensitivity. In: Flickinger MC, Drew SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation, vol 5. Wiley, New York, pp 2379–2406Google Scholar
  12. Chisti Y, Moo-Young M (1996) Bioprocess intensification through bioreactor engineering. Chem Eng Res Des 74A:575–583Google Scholar
  13. Corchero JL, Cubarsi R, Vila P, Aris A, Villaverde A (2001) Cell lysis in Escherichia coli cultures stimulates growth and biosynthesis of recombinant proteins in surviving cells. Microbiol Res 156:13–18CrossRefPubMedGoogle Scholar
  14. Cserjan-Puschmann M, Kramer W, Duerrschmid E, Striedner G, Bayer K (1999) Metabolic approaches for the optimisation of recombinant fermentation processes. Appl Microbiol Biotechnol 53:43–50CrossRefPubMedGoogle Scholar
  15. Curless C, Pope J, Tsai L (1990) Effect of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli. Biotechnol Prog 6:149–152CrossRefPubMedGoogle Scholar
  16. Dempster RP, Robinson CM, Harrison GBL (1996) Parasite vaccine development: large-scale recovery of immunogenic Taenis ovis fusion protein GST-45W(B/X) from Escherichia coli inclusion bodies. Parasitology Res 82:291–296CrossRefGoogle Scholar
  17. Doig SD, O’Sullivan LM, Patel S, Ward JM, Woodley JM (2001) Large scale production of cyclohexanone monooxygenase from Escherichia coli TOP10 pQR239. Enzyme Microb Technol 28:265–274CrossRefPubMedGoogle Scholar
  18. Ejiofor AO, Chisti Y, Moo-Young M (1996) Culture of Saccharomyces cerevisiae on hydrolysed waste cassava starch for production of baking-quality yeast. Enzyme Microb Technol 18:519–525CrossRefGoogle Scholar
  19. Guzman L-M, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130PubMedGoogle Scholar
  20. Heath DD, Jensen O, Lightowlers MW (2003) Progress in control of hydatidosis using vaccination—a review of formulation and delivery of the vaccine and recommendations for practical use in control programmes. Acta Tropica 85:133–143CrossRefPubMedGoogle Scholar
  21. Hellmuth K, Korz DJ, Sanders EA, Deckwer W-D (1994) Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1. J Biotechnol 32:289–298CrossRefPubMedGoogle Scholar
  22. Hoffmann F, Posten C, Rinas U (2001) Kinetic model of in vivo folding and inclusion body formation in recombinant Escherichia coli. Biotechnol Bioeng 72:315–322CrossRefPubMedGoogle Scholar
  23. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) Bergey’s manual of determinative bacteriology, 9th edn. Williams & Wilkins, BaltimoreGoogle Scholar
  24. Jeong KJ, Lee SY (1999) High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification. Appl Environ Microbiol 65:3027–3032PubMedGoogle Scholar
  25. Kleman GL, Strohl WR (1994) Acetate metabolism by Escherichia coli in high-cell-density fermentation. Appl Environ Microbiol 60:3952–3958PubMedGoogle Scholar
  26. Konz JO, King J, Conney CL (1998) Effect of oxygen on recombinant protein expression. Biotechnol Prog 14:393–409CrossRefPubMedGoogle Scholar
  27. Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer W-D (1995) Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol 39:59–65CrossRefPubMedGoogle Scholar
  28. Kweon D-H, Han NS, Park K-M, Seo J-H (2001) Overproduction of Phytolacca insularis protein in batch and fed-batch culture of recombinant Escherichia coli. Proc Biochem 36:537–542CrossRefGoogle Scholar
  29. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  30. Landwall P, Holme T (1997) Influence of glucose and dissolved oxygen concentrations on yields of Escherichia coli in dialysis culture. J Gen Microbiol 103:353–258Google Scholar
  31. Lee J, Lee SY, Park S (1997) Fed-batch culture of Escherichia coli w by exponential feeding of sucrose as a carbon source. Biotechnol Techniq 11:59–62CrossRefGoogle Scholar
  32. Li N, Qu L-J, Liu Y, Li Q, Gu H, Chen Z (1999) The refolding, purification, and activity analysis of a rice Bowman-Birk inhibitor expressed in Escherichia coli. Protein Express Pur 15:99–104CrossRefGoogle Scholar
  33. Lightowlers MW, Lawrence SB, Gaucci CG, Young J, Ralston MJ, Maas D, Heath DD (1996) Vaccination against hydatidosis using a defined recombinant antigen. Parasite Immunol 18:457–462CrossRefPubMedGoogle Scholar
  34. Lilie H, Schwarz E, Rudolph R (1998) Advances in refolding of proteins produced in E. coli. Curr Opin Biotechnol 9:497–501CrossRefPubMedGoogle Scholar
  35. Lim H-K, Jung K-H (1998) Improvement of heterologous protein productivity by controlling postinduction specific growth rate in recombinant Escherichia coli under control of the PL promoter. Biotechnol Prog 14:548–553CrossRefPubMedGoogle Scholar
  36. Lim H-K, Jung K-H, Park D-H, Chung S-I (2000) Production characteristics of interferon-a using an l-arabinose promoter system in a high-cell-density culture. Appl Microbiol Biotechnol 53:201–208CrossRefPubMedGoogle Scholar
  37. Madurawe RD, Chase TE, Tsao EI, Bentley WE (2000) A recombinant lipoprotein antigen against Lyme disease expressed in E. coli: fermentor operating strategies for improved yield. Biotechnol Progress 16:571–576CrossRefGoogle Scholar
  38. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538PubMedGoogle Scholar
  39. Middelberg APJ (2002) Preparative protein refolding. Trends Biotechnol 20:437–443CrossRefPubMedGoogle Scholar
  40. Oh G, Moo-Young M, Chisti Y (1998) Automated fed-batch culture of recombinant Saccharomyces cerevisiae based on on-line monitored maximum substrate uptake rate. Biochem Eng J 1:211–217CrossRefGoogle Scholar
  41. Robbens J, Raeymaekers A, Steidler L, Fiers W, Remaut E (1995) Production of soluble and active recombinant murine interleukin-2 in Escherichia coli: high level expression, kil-induced release, and purification. Protein Express Pur 6:481–486CrossRefGoogle Scholar
  42. Saluta M, Bell PA (1998) Troubleshooting GST fusion protein expression in E. coli. Life Science News 1:1–3Google Scholar
  43. Warnes A, Stephenson JR, Fooks AR, Melling J (1991) Expression of recombinant protein A from the lac promoter in Escherichia coli JM83 is not subject to catabolite repression when grown under specific conditions of continuous culture. Biotechnol Bioeng 38:1050–1058CrossRefGoogle Scholar
  44. Xu B, Jahic M, Blomsten G, Enfors S-O (1999) Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli. Appl Microbiol Biotechnol 51:564–571CrossRefPubMedGoogle Scholar
  45. Yee L, Blanch HW (1993) Recombinant trypsin production in high cell density fed-batch cultures of Escherichia coli. Biotechnol Bioeng 41:781–790CrossRefGoogle Scholar
  46. Zanette D, Dundon W, Soffientini A, Sottani C, Marinelli F, Akeson A, Sarubbi E (1998) Human IL-1 receptor antagonist from Escherichia coli: large-scale microbial growth and protein purification. J Biotechnol 64:187–196CrossRefPubMedGoogle Scholar
  47. Zhang J, Greasham R (1999) Chemically defined media for commercial fermentations. Appl Microbiol Biotechnol 51:407–421CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2005

Authors and Affiliations

  • Daniel Manderson
    • 1
  • Robert Dempster
    • 2
  • Yusuf Chisti
    • 1
    Email author
  1. 1.Institute of Technology and Engineering PN456Massey UniversityPalmerston NorthNew Zealand
  2. 2.AgResearch LtdWallaceville Animal Research CenterUpper HuttNew Zealand

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