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Application of the biotin-labeled toxin mutant for affinity isolation of associated proteins in the mammalian cells
- Yoon, Ju-Gyeong, Hwang, Hye-Jeong, Cho, Jin Ah
- Journal of bioscience and bioengineering 2018 v.125 no.5 pp. 497-504
- Vibrio cholerae, amino acids, bioengineering, biotin, biotinylation, cholera toxin, cytosol, diarrhea, endoplasmic reticulum, epithelial cells, epithelium, intestines, mammals, models, mutants, peptides, proteins, toxicity
- Cholera toxin (CT), one of the AB5 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.