Effect of Cytoplasmic Chaperons Of Soluble Expression Of Anti EGF-r Single Chain Antibody in E. coli

Nazari, Atefeh and Atefeh, Saber and Farajnia, Safar and latifi, Saeed (2017) Effect of Cytoplasmic Chaperons Of Soluble Expression Of Anti EGF-r Single Chain Antibody in E. coli. Masters thesis, University of Mohaghegh Ardabili.

Text (بررسي اثر چاپرونهاي سیتوپلاسمي در بيان محلول آنتي بادي تك زنجيره اي ضد EGFR در E. coli) Atefeh Nazari.pdf Download (1MB)

Abstract

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, multiplication and differentiation. Over expression of EGFR is associated with carcinogenesis that seen in more than 70% of head and neck cancers. Use of monoclonal antibodies to target EGFR blocks the EGFR downstream signaling pathway and results in suppression of tumor cells growth. E. coli is the preferred system for expression of single chain antibodies, but the advantages of this system affected by formation of inclusion bodies. Several strategies have been suggested to solve this problem among them utilization of molecular chaperones is one of the noticeable methods. In this study we tried to increase the soluble expression of huscfv antibody by co-expression with cytoplasmic chaperones. To achieve this purpose, chaperones plasmids of PG-tf2 (GroES- GroEL- tig), ptf16 (tig), pGro7 (GroES- GroEL), pKJE7(grp E- dnaJ- dnaK) and pG-KJE8 (groES- groEL- dnaK- dnaJ - grpE) were co-expressed with anti-EGFR hscFv construct in E. coli. Different concentrations of IPTG, temperatures and incubations times were used to achieve best results. Results of chaperones co-expression on soluble expression were analyzed by SDS-PAGE. Soluble hscFv was purified by Ni-NTA column, and then the biologic activity of the recombinant protein was determined by ELISA. the results of this study indicated that the highest concentrations of anti-EGFR hscFv were obtained by co-expressing chaperones pG-KJE8 and hscFv under special condition including 0.2 mM concentration of inducer (IPTG), culture temperature of 25 °C and 4h incubation time after induction. Keywords: EGFR, Inclusion body, Molecular chaperones, ScFv, Soluble expression

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