Mapping of the entomocidal fragment of Spodoptera-specific Bacillus thuringiensis toxin CryIC.
Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI proteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins.
Strizhov
N
N
Max-Planck Institut für Züchtungsforschung, Köln, Germany.
Keller
M
M
Koncz-Kálmán
Z
Z
Regev
A
A
Sneh
B
B
Schell
J
J
Koncz
C
C
Zilberstein
A
A
Konez-Kálmán
Z
Z
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D003001:Cloning, Molecular; D016384:Consensus Sequence; D010450:Endopeptidases / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D005798:Genes, Bacterial; D006460:Hemolysin Proteins; D007306:Insecticides / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D018411:Spodoptera / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010450:Endopeptidases; D014357:Trypsin