AU - Akbari, Bahman TI - Optimization of the Refolding Process for Recombinant Anti-EGFR Immunotoxin Produced in the Escherichia coli PT - JOURNAL ARTICLE TA - sjimu JN - sjimu VO - 29 VI - 5 IP - 5 4099 - http://sjimu.medilam.ac.ir/article-1-7119-en.html 4100 - http://sjimu.medilam.ac.ir/article-1-7119-en.pdf SO - sjimu 5 ABĀ  - Introduction: Overexpression of the EGFR is associated with carcinogenesis, and it is observed in more than 70% of head and neck cancers. The expression of an immunotoxin against EGFR designed as an alternative to full antibody led to the production of aggregated protein in the form of inclusion bodies. This study aimed to investigate the 8M urea and 6M guanidine hydrochloride approaches for obtaining the immunotoxin as the soluble and effective form with correct folding. Material & Methods: The BL21 (DE3) cells containing the pET28a-huimmunotoxin construct were induced by 1 mM IPTG at 37°C for 24 h, and the amount of expression was checked by SDS-PAGE. This immunotoxin was in the form of inclusion bodies and was solubilized individually in 8 M urea and 6 M guanidine hydrochloride and then purified by Ni-NTA affinity chromatography, which was observed as a single band in SDS-PAGE analysis. To correctly refold the obtained immunotoxin, the purified samples were poured into a dialysis bag, and denaturing agents were removed in a multi-step process called stepwise dialysis. The reactivity assessment of the purified and refold immunotoxin was assessed by ELISA technique using A431 cell lysate. Findings: The immunotoxin (17 mg/ml) was expressed using the bacteria cells in the form of inclusion bodies. The refolded humanized immunotoxin had a high reactivity with A431 cells, indicating the suitable folding of the purified immunotoxin. The 50% binding activity rates of humanized immunotoxin obtained from urea and guanidine hydrochloride approaches were 0.8 and 1.7 µg/ml, respectively. Discussion & Conclusion: The results of this study revealed that the urea approach was very effective in solubilizing and proper refolding of immunotoxins that were expressed in bacteria cells as inclusion bodies CP - IRAN IN - Dept of Medical Biotechnology, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran LG - eng PB - sjimu PG - 63 PT - Research YR - 2021