Expression and purification of hemolysin BL from local isolate of Bacillus cereus

  • Chanpen Wiwat and Raweewan Thiramana
Keywords: Bacillus cereus, Hemolysin BL, Enterotoxin, Food Poisoning

Abstract

Hemolysin BL (HBL) is the multi-component enterotoxin causing diarrheal type of Bacillus cereus food poisoning. B. cereus A6, previously isolated from sweet potato in Thailand and reported the presence of hblA and hblD genes by polymerase chain reaction (PCR), was used for hbl gene cloning in this study. Complete nucleotide sequence of B. cereus A6 hbl operon was achieved by primer walking technique. The hbl operon of B. cereus A6 divided into 4 coding sequences (CDS) identified as hblC, hblD, hblA and hblB genes consisting of 1,319, 1,221, 1,128 and 1,401 nucleotides encoding proteins of 439, 406, 375 and 466 amino acids with predicted sizes of 49.3, 43.8, 41.7 and 52.3 kDa, respectively. Both nucleotide and deduced amino acid sequences of B. cereus A6 hbl operon showed 98% sequence identities with those from B. cereus ATCC 14579 and B. thuringiensis. Subsequently, the recombinant plasmids containing each CDS of hblC, hblD and hblA were transformed and expressed in Escherichia coli BL21 (DE3). The recombinant His-tagged hblD and His-tagged hblA fusion proteins migrated at about 39.6 and 42.0 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were detected with anti-His antibody and could be purified by Ni-IDA column. The recombinant hblC protein migrated at about 42.8 kDa was not detected with anti-His antibody and could not bind to Ni-IDA column, but its cell lysate showed positive agglutination at titer higher than 128 with Bacillus cereus Enterotoxin-Reverse Passive Latex Agglutination (BCET-RPLA) test kit.

References

1. Kramer JM, Gilbert RJ. Bacillus cereus and other Bacillus species. In: Doyle MP, editor. Foodbome Bacterial Pathogens, New York : Marcel Dekke; 1989. p. 21-70.
2. Pirttijarvi TSM, Andersson MA, Salkinoja-Salonen MS. Properties of Bacillus cereus and other bacilli contaminating biomaterialbased industrial processes. Int J Food Microbiol 2000;60:231-9.
3. Granum PE, Lund T. Bacillus cereus and its food poisoning toxins. FEMS Microbiol Lett 1997;157:23-228.
Published
2017-06-22