Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties.
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This investigation was supported by 863 grants (2006AA02Z187, 2006AA10A212), SRFDP grant (20060542006), and NSFC (30670052) from China.
Zujiao Fu and Yunjun Sun contributed equally to this work.
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The full list of identified proteins and their internal tryptic peptides from strains HD1 and 4Q2-72 (DOC 102 KB)
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Fu, Z., Sun, Y., Xia, L. et al. Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry. Appl Microbiol Biotechnol 79, 875–880 (2008). https://doi.org/10.1007/s00253-008-1488-0
- Bacillus thuringiensis
- Polyacrylamide gel block
- Protoxin expression profile
- Insecticidal activity