A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium 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.

Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production

“…However, Luo et al (1997) and Rimler (2001) mentioned that this chromatography process results in a change of the outer membrane structure of the recombinant protein and affects its immunogenicity. To overcome this issue, many researchers have combined affinity chromatography with a purification method based on electroelution to avoid contamination by other proteins (Saraswat et al 2013;Vázquez-Iglesias et al 2017). In this study, electroelution was used to purify the rCP-SCMV from cell lysate to protect the immunogenicity of the protein and prevent the existence of contaminants.…”

“…Conformational changes in virus epitopes may arise during purification, as monitored in grapevine virus B and partial degradation of antigens due to proteolysis during the storage of sugar beets, which can lead to low immunogenicity of the antiserum and subsequently affects serological detection of these viruses (Abdel-Salam et al 2014), the proper antigen must be a highly purified protein with almost zero contaminant (≤ 1%). A purification technique using electroelution avoids contamination by other proteins that could appear during other protein purification techniques such as chromatography (Saraswat et al 2013;Vázquez-Iglesias et al 2017). The purified expressed plant viral protein as a recombinant fusion protein has been used as an antigen for raising virus-specific antibodies for immunodiagnosis.…”

PURIFIKASI PROTEIN IMUNOGENIK 31 DAN 56 kDa DARI KELENJAR SALIVA <em>Aedes aegypti</em>

“…Purifikasi protein imunogenik 31 dan 56 kDa dilakukan dengan metode elektroelusi dan dialisis. Metode elektroelusi bertujuan untuk mengeluarkan protein target dari dalam gel poliakrilamid sehingga didapatkan protein murni yang diinginkan (Vázquez-Iglesias et al 2017). Potongan gel poliakrilamid yang didalamnya terdapat protein target ditempatkan ke dalam membran selofan yang berisi buffer elektroda.…”

Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein

“…Elution via passive diffusion and electroelution are frequently used strategies [15,16]. Electroelution is a purification method that prevents contamination by other proteins that could occur during other protein purification methods like chromatography [17,18]. However, the easiest way to release protein molecules from a gel matrix is by adding water (or a buffer) to a portion of the gel that contains the desired protein.…”