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Research in Pharmaceutical Sciences، جلد ۱۴، شماره ۶، صفحات ۵۶۶-۵۷۳
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عنوان انگلیسی Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein
چکیده انگلیسی مقاله Recombinant epithelial cell adhesion molecule extracellular domain (EpEX) has a high potential as a candidate for passive and active immunotherapy as well as cancer vaccination. In the present study, EpEX was expressed as a thioredoxin fusion protein in Escherichia coli ( E. coli ). The effect of different hosts and expression conditions on the expression level of the fusion protein was also evaluated.Moreover, the effect of temperature and isopropyl-β-d-thiogalactopyranoside (IPTG) concentration on protein solubility was assessed. The codon optimized-synthetic gene was cloned into pET32a (+) expression vector and transformed into E. coli BL21 (DE3), Rosetta TM (DE3), and Origami TM (DE3).The protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Lowering the expression temperature to 16 °C and IPTG concentration to 0.5 mM also dramatically increased the volumetric productivity of the fusion protein. In optimum culture condition, high-level expression of the target fusion protein was detected in Rosetta TM (DE3) and Origami TM (DE3) (207 and 334 μg/mL, respectively), though they were expressed as inclusion bodies. No improvement was observed in the solubility of the fusion protein by reducing the temperature or IPTG concentration even when expressed in a TrxB/gor mutant strain. Results showed that Trx tag combined with other strategies utilized here could be effective to achieve high level of protein production but not effective in solubility improvement. However, new approaches might be necessary to enhance the solubility of EpEX in the E. coli system.
کلیدواژه‌های انگلیسی مقاله EpCAM,EpEX,Escherichia coli,Expression,Thioredoxin.
نویسندگان مقاله | Farideh Rasooli
1Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran. 2Protein Technology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran.


| Atieh Hashemi


نشانی اینترنتی http://rps.mui.ac.ir/index.php/jrps/article/view/1951
فایل مقاله اشکال در دسترسی به فایل - ./files/site1/rds_journals/115/article-115-2233607.pdf
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زبان مقاله منتشر شده en
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نوع مقاله منتشر شده Original Article
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