Rapid Purification of Endotoxin-Free RTX Toxins
AbstractCytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA). View Full-Text
- Supplementary File 1:
PDF-Document (PDF, 494 KB)
Share & Cite This Article
Stanek, O.; Masin, J.; Osicka, R.; Jurnecka, D.; Osickova, A.; Sebo, P. Rapid Purification of Endotoxin-Free RTX Toxins. Toxins 2019, 11, 336.
Stanek O, Masin J, Osicka R, Jurnecka D, Osickova A, Sebo P. Rapid Purification of Endotoxin-Free RTX Toxins. Toxins. 2019; 11(6):336.Chicago/Turabian Style
Stanek, Ondrej; Masin, Jiri; Osicka, Radim; Jurnecka, David; Osickova, Adriana; Sebo, Peter. 2019. "Rapid Purification of Endotoxin-Free RTX Toxins." Toxins 11, no. 6: 336.
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.