Rapid Purification of Endotoxin-Free RTX Toxins

Abstract

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).

Production of Highly Active Recombinant Dermonecrotic Toxin of Bordetella Pertussis

“…High level production of soluble recombinant DNT protein was then achieved by expression of the dnt gene at 20 °C for 24 h in Escherichia coli Rosetta 2 cells that harbor tRNAs genes for rare codons and efficiently translate the mRNA transcripts of G:C-rich genes. The C-terminally tagged DNT protein was purified from the cytosolic fraction of bacterial lysates by a combination of pre-purification on DEAE Sepharose at pH 7.4 followed by ion metal affinity chromatography on Ni-NTA agarose ( Figure 1 b), where the contaminating E. coli outer membrane lipopolysaccharide (LPS) and other components were removed by extensive washing of the column with 1% ( v / v ) Triton X-100 [ 31 ]. The yield after the two purification steps was 3 mg of LPS-free (<100 EU/mg) DNT per 1 L of bacterial culture ( Figure 1 c).…”

RTX-Toxins

“…For this, the CyaA has to be detoxified by the insertion of foreign antigenic determinants into the adenylyl cyclase domain [29]. A severe problem for the use of recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli is the presence of high amounts of E. coli lipopolysaccharide (LPS or endotoxin) [29,30]. A contribution to this Special Issue describes a simple procedure for the purification of RTX cytolysins from LPS [30].…”

“…A severe problem for the use of recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli is the presence of high amounts of E. coli lipopolysaccharide (LPS or endotoxin) [29,30]. A contribution to this Special Issue describes a simple procedure for the purification of RTX cytolysins from LPS [30]. This procedure is based on the complete unfolding of the toxins in 8 M urea, followed by their binding to a chromatographic medium.…”

“…This procedure is based on the complete unfolding of the toxins in 8 M urea, followed by their binding to a chromatographic medium. Contaminating LPS is removed by an extensive wash of the column with urea and detergent [30]. Finally, residual detergent is removed by a wash with 8 M urea, and the RTX protein can be eluted from the column.…”

“…Finally, residual detergent is removed by a wash with 8 M urea, and the RTX protein can be eluted from the column. The authors of the study describe the application of this method to four RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA) [30].…”