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Iranian Journal of Biotechnology، جلد ۱۸، شماره ۲، صفحات ۶۶-۷۳
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عنوان انگلیسی Cytoplasmic Chaperones Enhance Soluble Expression of Anti-EGFR huscFv in Escherichia coli
چکیده انگلیسی مقاله Background: Overexpression of EGFR is associated with carcinogenesis in more than 70% of head and neck cancers. Anti-EGFR monoclonal antibodies bind to the extracellular domain of EGFR and block the EGFR downstream signaling pathway, which results in the suppression of the growth of the tumor cells. Escherichia coli is the preferred system for expressing various recombinant proteins, including single chain antibodies, but the formation of inclusion bodies negatively affects the efficacy of this system. Several strategies have been suggested to solve this problem, notably the utilization of molecular chaperones.
Objectives: In this study, we attempted to increase the soluble expression of huscfv antibody via co-expression with the cytoplasmic chaperones.
Materials and Methods: To achieve this purpose, chaperones plasmids pG-KJE8, pGro7, pKjE7, pTf16 and pG-Tf2 encoding cytoplasmic chaperones were co-expressed with the humanized anti-EGFR scFv construct in E. coli. Different temperatures, incubations times, and concentrations of IPTG were used to produce an active antibody with the highest solubility. Results were analyzed by SDS-PAGE. Soluble huscFv was purified by Ni-NTA column and the biologic activity of the recombinant protein was determined by ELISA.
Result: The results indicated that the highest concentrations of humanized anti-EGFR scFv were obtained by co-expression of huscFv via chaperone plasmid pG-KJE8 with 0.2 mM concentration of inducer (IPTG), culture temperature of 25 °C, and 4 h incubation time after induction.
Conclusion: In conclusion, co-expression with chaperones could be used as an efficient strategy to produce soluble active ScFvs in E. coli.
کلیدواژه‌های انگلیسی مقاله EGFR,Inclusion body,Molecular chaperones,scFv,soluble expression
نویسندگان مقاله Atefeh Nazari |
Department of Biology, University of Mohaghegh Ardebili, Ardebil, Iran

Safar Farajnia |
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Saber Zahri |
Department of Biology, University of Mohaghegh Ardebili, Ardebil, Iran

Nazanin Bagherlou |
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Asghar Tanoumand |
Maragheh University of Medical Sciences, Maragheh, Iran

Leila Rahbarnia |
Infectious and tropical diseases research center, Tabriz University of Medical Sciences, Tabriz, Iran

نشانی اینترنتی https://www.ijbiotech.com/article_114423_b5b49e536bda8655414c7a6183d0a958.pdf
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