چاپ صفحه |  صفحه نخست سامانه |  نویسندگان |  اطلاعات تفضیلی |  دانلود مقاله | علوم پزشکی کرمانشاه |
| نویسنده | نفر چندم مقاله |
|---|---|
| بهمن اکبری | اول |
| عنوان | متن |
|---|---|
| کلمات کلیدی | Guanidine hydrochloride, Inclusion body, Refolding, Urea. |
| چکیده | Abstract Background: High-level expression of many recombinant proteins in E. coli leads to formation of inclusion bodies, highly aggregated proteins; they can also form in mammalian, insect, and yeast cells. Over expression of a humanized immunotoxin (IT) against epidermal growth factor receptor (EGFR) led to the production of inclusion bodies. The aim of this study was optimization of refolding process for recombinant humanized anti-EGFR IT produced in the E. coli. Methods: The BL21 (Plys S) cells containing a pET-22b- humanized anti-EGFR IT construct constructed and characterized in previous work- were induced by 0.25mmol/l IPTG at 25 C° for 4 h and the amount of expression was checked by SDS-PAGE, even though the majority of expressed proteins appeared as inclusion bodies. These inclusion bodies were individually solubilized in 8 M urea and 6 M guanidine hydrochloride and then purified by Ni-NTA affinity chromatography which was seen as a single band in SDS-PAGE analysis. Results: The proper refolding humanized IT achieved by stepwise dialysis method. Reactivity assessment of humanized IT obtained from the urea approach with A431 carcinoma cells by relative ELISA test revealed that the refolded humanized IT had a high reactivity, indicating suitable folding of purified antibody. The 50% binding activity of humanized IT achieved from urea and guanidine hydrochloride approaches were 0.5 and 1.75 µg/ml concentrations, respectively. Conclusion: The results of this study revealed that the urea approach was very effective in solubilizing and refolding of IT that expressed in bacteria cells as inclusion bodies. |
| متن مقاله | Abstract Background: High-level expression of many recombinant proteins in E. coli leads to formation of inclusion bodies, highly aggregated proteins; they can also form in mammalian, insect, and yeast cells. Over expression of a humanized immunotoxin (IT) against epidermal growth factor receptor (EGFR) led to the production of inclusion bodies. The aim of this study was optimization of refolding process for recombinant humanized anti-EGFR IT produced in the E. coli. Methods: The BL21 (Plys S) cells containing a pET-22b- humanized anti-EGFR IT construct constructed and characterized in previous work- were induced by 0.25mmol/l IPTG at 25 C° for 4 h and the amount of expression was checked by SDS-PAGE, even though the majority of expressed proteins appeared as inclusion bodies. These inclusion bodies were individually solubilized in 8 M urea and 6 M guanidine hydrochloride and then purified by Ni-NTA affinity chromatography which was seen as a single band in SDS-PAGE analysis. Results: The proper refolding humanized IT achieved by stepwise dialysis method. Reactivity assessment of humanized IT obtained from the urea approach with A431 carcinoma cells by relative ELISA test revealed that the refolded humanized IT had a high reactivity, indicating suitable folding of purified antibody. The 50% binding activity of humanized IT achieved from urea and guanidine hydrochloride approaches were 0.5 and 1.75 µg/ml concentrations, respectively. Conclusion: The results of this study revealed that the urea approach was very effective in solubilizing and refolding of IT that expressed in bacteria cells as inclusion bodies. |
| نتیجه مقاله | Results: The proper refolding humanized IT achieved by stepwise dialysis method. Reactivity assessment of humanized IT obtained from the urea approach with A431 carcinoma cells by relative ELISA test revealed that the refolded humanized IT had a high reactivity, indicating suitable folding of purified antibody. The 50% binding activity of humanized IT achieved from urea and guanidine hydrochloride approaches were 0.5 and 1.75 µg/ml concentrations, respectively. Conclusion: The results of this study revealed that the urea approach was very effective in solubilizing and refolding of IT that expressed in bacteria cells as inclusion bodies. |
| نام فایل | تاریخ درج فایل | اندازه فایل | دانلود |
|---|---|---|---|
| Oral Certificate(s).pdf | 1397/03/21 | 1322872 | دانلود |
| article.jpg | 1397/03/21 | 132883 | دانلود |
| - Copy.png | 1397/03/21 | 78215 | دانلود |
