A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum

Abstract

Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

Keywords: Alpha-toxin; Antibodies; Clostridium septicum; Electroelution; Slot blot.

Figure 1. Protein profile of Clostridium septicum extracellular medium.

(A) SDS-PAGE analysis by Coomassie Brilliant Blue R-250 stained. (B) Non-denaturing Native-PAGE analysis by Coomassie Brilliant Blue R-250 stained. Protein bands 1–17 were cut and analyzed by MS/MS. kDa indicates migration of the protein markers.

Figure 2. Western blot analysis of the Clostridium septicum extracellular medium using our anti-Clostridium septicum alpha toxin and the Clostridium septicum (Gas-Gangrene) antitoxin (NIBSC code: VI).

(A) SDS-PAGE and western blot analysis using anti-C. septicum alpha toxin. (B) Native-PAGE and western blot analysis using anti-C. septicum alpha toxin. (C) SDS-PAGE and western blot analysis using the C. septicum (Gas-Gangrene) antitoxin (NIBSC code: VI). kDa indicates migration of the protein markers. The arrows indicate alpha toxin. A total of 3 μg of total protein per lane were loaded.

Figure 3. Analysis of the specificity of the anti-Clostridium septicum alpha toxin by western blotting with other Clostridium spp.

Lane 1 extracellular medium of C. septicum Lane 2 extracellular medium of C. perfringens type D, Lane 3 extracellular medium of C. haemolyticum, Lane 4 extracellular medium of C. tetani and Lane 5 extracellular medium of C. sordellii. Lane M indicates migration of protein molecular weight markers (kDa). The arrows indicate alpha toxin. A total of 3 μg of total protein per lane were loaded.

Figure 4. Slot blot analysis for the quantification of different concentrations of purified alpha toxin.

(A) Several concentrations of purified alpha toxin were analyzed using the protocol detail in ‘Material and Methods’. The culture medium and PBS serve as a negative control. (B) This figure shows the linear relationship between optical density and protein concentration gave a correlation coefficient of 0.9931. (C) The logarithm concentration of the alpha toxin was plotted on x axis and the O.D. values were plotted in y axis. The 4-parameter logistic (4PL) model was used for the calibration curve. A very good fitting, R² = 0.995, was demonstrated.