Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 25, Issue 3, Pages 465-471Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S1046-5928(02)00037-2
Keywords
RTX toxin; type I secretion; HlyA; LktA; localized juvenile periodontitis
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Funding
- NIGMS NIH HHS [R01 GM 29085] Funding Source: Medline
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The RTX (repeats in toxin) family of toxins is important in the pathogenesis of many Gram-negative bacteria. The oral and systemic human pathogen Actinobacillus actinomycetemcomitans produces a member of this family known as leukotoxin (LtxA). Previously, we found that LtxA is secreted into culture supernatants of A. actinomycetemcomitans and that this protein is abundant and relatively pure. Here, we report a large-scale method for the isolation and purification of LtxA from culture supernatants of A. actinonlycetoncomitans strain JP2, The purification scheme involves ammonium sulfate precipitation of culture supernatants, dialysis, and ultrafiltration to concentrate LtxA to approximately 10mg/ml. We found that LtxA remained soluble in buffer that contained at least 250 mM NaCl. Purified LtxA was >98%, pure and the final preparations were active against HL-60 cells. The entire purification protocol can be completed within 2 days. The ability to readily obtain a large amount of purified leukotoxin should accelerate investigations into the structure and biology of this important virulence factor. (C) 2002 Elsevier Science (USA). All rights reserved.
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