Efficient and Scalable Process to Produce Novel and Highly Bioactive Purified Cytosolic Crystals from Bacillus thuringiensis

Jeffrey Chicca, Nicholas R. Cazeault, Florentina Rus, Ambily Abraham, Carli Garceau, Hanchen Li, Samar M. Atwa, Kelly Flanagan, Ernesto R. Soto, Mary S. Morrison, David Gazzola, Yan Hu, David R. Liu, Martin K. Nielsen, Joseph F. Urban, Gary R. Ostroff, Raffi V. Aroian

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins.

Original languageEnglish
JournalMicrobiology spectrum
Issue number4
StatePublished - Aug 2022

Bibliographical note

Funding Information:
This work was financially supported by the National Institutes of Health National Institute of Allergy and Infectious Diseases grants R01-AI056189 and R01-AI150866 to R.V.A. and Agriculture and Food Research Initiative Competitive Grant 2015-11323 from the USDA National Institute of Food and Agriculture to R.V.A.

Publisher Copyright:
© 2022 Chicca et al.


  • Bacillus thuringiensis
  • anthelmintic
  • crystal protein
  • drugs
  • parasites
  • purification

ASJC Scopus subject areas

  • Physiology
  • Ecology
  • Immunology and Microbiology (all)
  • Genetics
  • Microbiology (medical)
  • Cell Biology
  • Infectious Diseases


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