ISOLATION OF LYSOZYME LIKE PROTEIN FROM CROCODILE LEUKOCYTE EXTRACT (Crocodylus siamensis)
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Abstract
In mammals, white blood cells (neutrophils, macrophages and natural killer cells) contain components of immune response such as antimicrobial proteins and antimicrobial peptides. A variety of proteins and peptides are effecter molecules able to kill microbial pathogens. This study aims to screen and purify antimicrobial substances from crocodile leukocyte extract. The crude leukocyte extract showed antibacterial activity toward Psudomonas aeruginosa, Psudomonas aeruginosa ATCC 2785, Salmonella typhi, Salmonella typhi B, Salmonella typhi ATCC 5784, Vibrio cholerae, Bacillus megaterium, Staphylococus epidermidis and Candida albicans. The crude extract was purified by anion exchange column chromatography (FPLC) and 4 protein peaks (P1-P4) were obtained. Protein fractions (P1-P3) showed antibacterial and antifungal activity like the crude leukocyte extract. Protein fraction (P4) displayed specific activity against to Pseudomonas aeruginosa ATCC 2785 and Vibrio cholerae. The leukocyte extract and protein fraction (P1) were characterized by zymogram refolding gel. Leukocyte extract demonstrated clear zones of inhibition at band molecular weight about 21 and 15 kDa. Antimicrobial activity and MW of these proteins are similar to that of lysozyme. When determined by western immuno blotting using anti-hen lysozyme as primary antibody, the protein of MW at 14-15 kDa could cross react with anti-hen lysozyme. In addition, the result from MALDI-TOF MS-MS confirmed that the 14-15 kDa protein was lysozyme. This result indicated that antimicrobial substance in crocodile leukocyte extract was lysozyme-like protein.
Keywords: antimicrobial protein, white blood cell, Crocodylus siamensis
Corresponding author: E-mail: [email protected]
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