Publication: BACILLUS THURINGIENSIS 18 (Bt18) PARASPORAL PROTEINS WITH ANTI LEUKAEMIC ACTIVITY: PURIFICATION, CHARACTERISATION AND IMMUNOASSAY
Date
2008-06
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Publisher
International Medical University
Abstract
To achieve the objectives, the following was performed and analysed. Bt18 parasporal proteins were separated through anion exchange chromatography and a 68-kDa parasporal protein with cytotoxic activity was purified. Polyclonal IgG (anti-Bt18) for the 68-kDa parasporal protein was successfully raised and purified. The purified anti-Bt-18 IgG showed interaction with parasporal proteins of Bt18, using immunoblot analysis. The purified anti-Bt18 IgG was tested for cross-reactivity against other Bt and Bacillus isolates using Indirect ELISA (quantitative assay). It was observed that Bt isolates share similar antigenic properties with the 68-kDa protein of Bt18 whereas very low cross-reactivity was observed with Bacillus sphaericus, Bacillus subtilis and Bacillus cereus isolates (less than 17%). Immunoblot analysis (qualitative assay) for cross-reactivity also showed that there was a 68- kDa protein in Bt isolates. Cross-reactivity studies via quantitative and qualitative analysis was repeated using purified Bt parasporal protein. Indirect ELISA assay showed the percentage of cross-reactivity was reduced up to 60% after purification for some Bt isolates; Bt2, Bt10, Bt15, Bt19 and Bt20. Meanwhile other Bt isolates still showed more than 90% cross-reactivity. Qualitative assay for cross-reactivity via immunoblot assay detected bands for all the Bt isolates. Similar band profiles were observed in immunoblots using either purified or unpurified Bt parasporal proteins.
Receptor binding assay revealed that Bt18 parasporal proteins bound to a 34-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 34-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein with multiple functions, including a vital role in mitochondrial apoptosis. It is interesting to note that Bt18 produces parasporal proteins that act like parasporins. Binding of Bt18 parasporal proteins to GAPDH is a significant finding in this study as literature shows no studies reports on identification of a putative receptor for parasporins. Immuno-staining to detect the binding of Bt18 parasporal protein on CEM-SS cells showed brownish ring formation around the cells, suggesting that the protein may bind to a receptor on the plasma membrane of cells. The specificity of Bt18 parasporal proteins for different monosaccharides showed preferential binding (decreasing order) to D-mannose, D-Lactose and D-glucose. Glycoprotein staining showed that Bt18 parasporal proteins contain three distinct glycoprotein bands about; 200, 100 and 68-kDa. Therefore, two key conclusions can be made; that there is a target protein for Bt18 parasporal proteins in CEM-SS cells and that Bt18 parasporal proteins are lectins that specifically recognise monosaccharides.
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Keywords
Bacillus thuringiensis, Immunoassay, Glycoproteins