Rapid Purification of Endotoxin-Free RTX Toxins

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F19%3A00507897" target="_blank" >RIV/61388971:_____/19:00507897 - isvavai.cz</a>

Alternative codes found

RIV/00216208:11310/19:10396418

Result on the web

<a href="https://www.mdpi.com/2072-6651/11/6/336" target="_blank" >https://www.mdpi.com/2072-6651/11/6/336</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3390/toxins11060336" target="_blank" >10.3390/toxins11060336</a>

Result language

angličtina

Original language name

Rapid Purification of Endotoxin-Free RTX Toxins

Original language description

Cytolytic 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).

Czech name

—

Czech description

—

Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10606 - Microbiology

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2019

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Toxins

ISSN

2072-6651

e-ISSN

—

Volume of the periodical

11

Issue of the periodical within the volume

6

Country of publishing house

CH - SWITZERLAND

Number of pages

12

Pages from-to

336

UT code for WoS article

000475328000033

EID of the result in the Scopus database

2-s2.0-85068438043