The Bacillus thuringiensis Cry1Aa toxin: effects of trypsin and chymotrypsin site mutations on toxicity and stability.
The objective of the present work was to create an active Cry1Aa toxin showing enhanced resistance to degradation by spruce budworm (Choristoneura fumiferana) midgut proteases by mutating potential chymotrypsin and trypsin sites. Fourteen Cry1Aa mutants were created in an Escherichia coli-Bacillus shuttle vector and expressed in a crystal minus Bacillus thuringiensis host. Using spruce budworm gut juice, commercial bovine trypsin and chymotrypsin we performed protease resistance assays with Cry1Aa wild type and mutant toxins. Although many mutants showed little or no change, several mutants showed a > 2-fold increase (R543S, R566G, and F570S) up to a > 4-fold increase in toxicity (F576S), in bioassay studies against C. fumiferana. The in vitro protease resistance assay results indicated a possible involvement of other gut juice components in toxin overdigestion.
Bah
Aliou
A
Biotechnology Research Institute, National Research, Council, Montreal, Que., Canada H4P 2R2.
van Frankenhuyzen
Kees
K
Brousseau
Roland
R
Masson
Luke
L
D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins; D001681:Biological Assay / Q000379:methods; D001711:Biotransformation / Q000502:physiology*; D002918:Chymotrypsin / Q000378:metabolism*; D004064:Digestive System / Q000201:enzymology; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism*; D005750:Gastric Juice / Q000201:enzymology*; D006460:Hemolysin Proteins; D009036:Moths / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation / Q000502:physiology; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D002918:Chymotrypsin; D014357:Trypsin