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 In E. coli, high-level expression of many recombinant proteins leads to the formation of inclusion bodies, highly aggregated protein; however, they can also form in mammalian, insect, and yeast cells. Over expression of a humanized single chain variable fragment (huscFv) antibody against EGFR designed as an alternative to full length antibody led to the production of inclusion bodies. The aim of this study was optimization of refolding process for recombinant anti-EGFR huscFv produced in the E. coli. The BL21 (DE3) cells containing a pET28a-huscFv construct were induced by 1 mM IPTG at 37 C° for 20 h and the amount of expression was checked by SDS-PAGE, even though the majority of expressed proteins appeared as inclusion bodies. These his-tag fusion 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. The proper refolding humanized scFv achieved by stepwise dialysis method. Reactivity assessment of humanized scFv obtained from the urea approach with A431 carcinoma cells by relative ELISA test revealed that the refolded humanized scFv had a high reactivity, indicating suitable folding of purified antibody. The 50% binding activity of humanized scFv achieved from urea and guanidine hydrochloride approaches were 0.8 and 2.1 µg/ml concentrations, respectively. The results of this study revealed that the urea approach is very effective in solubilizing and refolding of scFvs that expressed in bacteria cells as inclusion bodies. |
| متن مقاله | Abstract In E. coli, high-level expression of many recombinant proteins leads to the formation of inclusion bodies, highly aggregated protein; however, they can also form in mammalian, insect, and yeast cells. Over expression of a humanized single chain variable fragment (huscFv) antibody against EGFR designed as an alternative to full length antibody led to the production of inclusion bodies. The aim of this study was optimization of refolding process for recombinant anti-EGFR huscFv produced in the E. coli. The BL21 (DE3) cells containing a pET28a-huscFv construct were induced by 1 mM IPTG at 37 C° for 20 h and the amount of expression was checked by SDS-PAGE, even though the majority of expressed proteins appeared as inclusion bodies. These his-tag fusion 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. The proper refolding humanized scFv achieved by stepwise dialysis method. Reactivity assessment of humanized scFv obtained from the urea approach with A431 carcinoma cells by relative ELISA test revealed that the refolded humanized scFv had a high reactivity, indicating suitable folding of purified antibody. The 50% binding activity of humanized scFv achieved from urea and guanidine hydrochloride approaches were 0.8 and 2.1 µg/ml concentrations, respectively. The results of this study revealed that the urea approach is very effective in solubilizing and refolding of scFvs that expressed in bacteria cells as inclusion bodies. |
| نتیجه مقاله | . The proper refolding humanized scFv achieved by stepwise dialysis method. Reactivity assessment of humanized scFv obtained from the urea approach with A431 carcinoma cells by relative ELISA test revealed that the refolded humanized scFv had a high reactivity, indicating suitable folding of purified antibody. The 50% binding activity of humanized scFv achieved from urea and guanidine hydrochloride approaches were 0.8 and 2.1 µg/ml concentrations, respectively. The results of this study revealed that the urea approach is very effective in solubilizing and refolding of scFvs that expressed in bacteria cells as inclusion bodies. |
| نام فایل | تاریخ درج فایل | اندازه فایل | دانلود |
|---|---|---|---|
| article scfv.pdf | 1397/03/21 | 226416 | دانلود |
| abstract book.pdf | 1397/03/21 | 4627281 | دانلود |
| img029.jpg | 1397/03/21 | 334767 | دانلود |
| img026.jpg | 1397/03/21 | 368337 | دانلود |
