Application of the biotin-labeled toxin mutant for affinity isolation of associated proteins in the mammalian cells

Cholera toxin (CT), one of the AB 5 bacterial toxin families, is produced by Vibrio cholerae , breeches the intestinal epithelial barrier and enters host epithelial cells to cause the massive secretory diarrhea. This study focused on understanding the retro-translocation machinery of the bacterial toxin using biotin-avidin technology to explain toxin trafficking from the endoplasmic reticulum (ER) to the cytosol. Because the association between the A1 chain of CT and other components of the retro-translocation machinery is likely transient or very weak, the successful bioengineering of such a mutant to be trapped as an intermediate in ER is essential for affinity isolation and further analysis. Here, we prepared a mutant toxin that 15 amino acid Biotin Acceptor Peptide (BAP) was fused to the C-terminal of A1 chain of CT. Biotinylation efficiency of the BAP-inserted cholera toxin (BT) was nearly 100%. Moreover, BT was functionally toxic and successfully pulled down by NeutrAvidin in vitro and in vivo. However, NeutrAvidin-bound biotinylated BT was not toxic. These results suggest the possibility of a plug effect of the biotin-NeutrAvidin-BT complex stuck in the ER without retro-translocation to the cytosol. Therefore, this model might identify the interacting proteins with A1 chain of CT in the host cells by holding the moment of retro-translocation of the bacterial toxin. In conclusion, this study established the model using biotin-avidin technology to elucidate the molecular basis for retro-translocation of bacterial toxin from within the lumen of ER to the cytosol.