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Protein Expr Purif. 2002 Aug;25(3):465-71.

Purification of secreted leukotoxin (LtxA) from Actinobacillus actinomycetemcomitans.

Author information

1
Department of Microbiology, College of Physicians and Surgeons, Columbia University, 701 W. 168th St., New York, NY 10032, USA. sk564@columbia.edu

Abstract

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. actinomycetemcomitans 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 250mM 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.

PMID:
12182827
DOI:
10.1016/s1046-5928(02)00037-2
[Indexed for MEDLINE]

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