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Scale-up of transgenic tobacco cells that express intimin of enterohemorrhagic Escherichia coli O157:H7 for use as a transitional platform for an oral cattle vaccine

  • Kristin M. O’Neill
  • Anne M. Schilthuis
  • Calvin A. Leiter
  • Kurt M. Neihaus
  • Nicole A. Judge
  • Edda Twiddy
  • Alison D. O’Brien
  • Wayne R. CurtisEmail author
Molecular Farming/Metabolic Engineering/Secondary Metabolism

Abstract

Intimin is the primary adhesin protein of enterohemorrhagic Escherichia coli O157:H7, a pathogen carried by cattle and transmitted to humans via contaminated food and water. This work describes the rapid production of 1.3 kg dry weight of transgenic tobacco cells expressing the C-terminal 261 amino acids of intimin (Int261) for subsequent oral vaccine testing to reduce cattle colonization by E. coli O157:H7. Both a stirred-tank bioreactor (60 L) and a less capital-intensive oxygenated carboy culture were shown feasible for generating this scale of transgenic plant tissue culture biomass. Antibiotic selection pressure was not required during scale-up to maintain Int261 expression over the 13-wk culture period. Extended medium autoclave times of up to 90 min used for bioreactor sterilization had only minimal impact on nutrient uptake, culture growth, and intimin expression. Plant tissue was transformed, produced in vitro, and available for feeding studies in a fraction of the time required to develop and grow transgenic plants.

Keywords

Plant tissue culture Protein expression Bioreactor operation Oxygen demand Bovine vaccine Pathogenic E. coli 

Notes

Acknowledgments

We would like to thank Aaron Raudabaugh and Mathew Murray for their assistance in carrying out the bioreactor runs, Brandon Curtis for the execution of the autoclaved medium experiment and general laboratory assistance, and Sergio Florez for the critical review of the manuscript. A.M.S. acknowledges summer research support from the Penn State’s Life Science Consortium, which provided support toward completion of an honors thesis in the Schreyer’s University Scholars Program (Growth of transgenic tobacco cell cultures for calf vaccination against pathogenic Escherichia coli, The Pennsylvania State University, BS Thesis, 50 p., May 2004). W.R.C. would like to acknowledge the National Science Foundation grant # BCS-0003926 and GOALI program, as well as grant # CBET-1035072, which has provided time to complete the publication process. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. A.O.B. acknowledges financial support from the National Institutes of Health (grant # AI 20148–20), and the American Meat Institute Foundation. Additionally, we thank the Boyce Thompson Institute for Plant Research (Ithaca, NY) for providing the NT-1 cells and the training N.A.J. received in plant cell transformation methods.

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Copyright information

© The Society for In Vitro Biology 2015

Authors and Affiliations

  • Kristin M. O’Neill
    • 1
    • 3
  • Anne M. Schilthuis
    • 1
  • Calvin A. Leiter
    • 1
  • Kurt M. Neihaus
    • 1
  • Nicole A. Judge
    • 2
  • Edda Twiddy
    • 2
  • Alison D. O’Brien
    • 2
  • Wayne R. Curtis
    • 1
    Email author
  1. 1.Department of Chemical EngineeringThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Department of Microbiology and Immunology, F. Edward Herbert School of MedicineUniformed Services University of the Health SciencesBethesdaUSA
  3. 3.Merck, Bioprocess DevelopmentKenilworthUSA

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