| Autor: |
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 |
| Rok vydání: |
2022 |
| Popis: |
Bacillus thuringiensis or Bt is a Gram-positive soil bacterium, widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops and vector diseases. 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, target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described IBaCC (for Inactivated Bacteria with Cytosolic Crystal(s)) 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 active pharmaceutical ingredient form of Bt Cry proteins.NON-TECHNICAL IMPORTANCE PARAGRAPHBacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies show they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called Purified Cytosolic Crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins are desirable and where these methods will find great utility. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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