A Cell-Permeable Fusion Protein Based on Clostridium botulinum C2 Toxin for Delivery of p53 Tumorsuppressor into Cancer Cells

Abstract

Genetically engineered bacterial protein toxins are attractive systems for delivery of exogenous proteins into the cytosol of mammalian cells. The binary C2 toxin from C. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component C2IIa and the genetically modified adaptor domain C2IN that act in concert to trigger cell uptake. The p53 tumor suppressor protein has a crucial function in suppressing carcinogenesis and is frequently inactivated by diverse mechanisms in human tumor cells. Therefore, we constructed a C2IN-p53 fusion protein, which is internalized into cancer cells by C2IIa. To this end, the C2IN-p53 fusion construct was overexpressed in E. coli with good solubility, purified by heparin affinity chromatography and protein identity was confirmed by immunoblotting. We demonstrated that the fusion protein is capable of binding to the p53 consensus-DNA with high affinity in a p53-specific manner in vitro. Next, the internalization of C2IN-p53 was monitored in HeLa cells by cell fractionation and immunoblot analysis, which revealed a C2IIa-mediated translocation of the fusion protein into the cytosol. The uptake was also shown in A549 and Saos-2 cells with similar efficiency. These findings were further corroborated by confocal immunofluorescence analyses of C2IN-p53/C2IIa-treated HeLa and A549 cells, displaying predominantly cytoplasmic localization of the fusion construct.

Chikungunya virus requires an intact microtubule network for efficient viral genome delivery

“…Subsequently, cells were washed thoroughly to remove unbound virus. In order to fractionate the cells, cells were permeabilized by incubation with 50μg/mL digitonin (Sigma-Aldrich) for 5 min at RT and subsequently for 30 min on ice [25]. Afterwards, the supernatant was carefully collected to obtain the cytosolic fraction and the remaining permeabilized cells were directly lysed in the cell culture plate to obtain the membrane fraction.…”

“…In these experiments, U-2 OS were used as these cells are slightly more permissive to infection than BS-C-1 cells. Cells were fractionated by use of digitonin treatment, which specifically permeabilizes the plasma membrane [25,29]. First, we checked the fractionation efficiency in four independent experiments by Western blot.…”

Chikungunya virus requires an intact microtubule network for efficient viral genome delivery

“…Subsequently, cells were washed thoroughly to remove unbound virus. In order to fractionate the cells, cells were permeabilized by incubation with 50µg/mL digitonin (Sigma-Aldrich) for 5 min at RT and subsequently for 30 min on ice [25]. Afterwards, the supernatant was carefully collected to obtain the cytosolic fraction and the remaining permeabilized cells were directly lysed in the cell culture plate to obtain the membrane fraction.…”

“…In these experiments, U-2 OS were used as these cells are slightly more permissive to infection than BS-C-1 cells. Cells were fractionated by use of digitonin treatment, which specifically permeabilizes the plasma membrane [25,28].…”

Engineering of bacterial toxins for research and medicine

“…The N-terminal domain of C2I (C2IN) binds to C2IIa and is crucial for C2IIa-mediated internalization, but does not comprise the enzyme domain that is responsible for cytotoxic effects. Hence, C2IN has been fused to the virulence factor SpvB from Salmonella enterica to trigger its internalization into mammalian cells [91], to the biotin-binding protein streptavidin, generating a delivery system for biotin-labeled molecules [92], and to the tumor suppressor protein p53 [93]. Also, the B chains of DT or Pseudomonas exotoxin A have been linked with the enzymatic component from ricin and the resulting chimeric toxins efficiently translocate the ricin enzymatic moiety [94,95].…”