Effective Strategies to Overcome the Insolubility of Recombinant ScFv Antibody against EpCAM Extracellular Domain in E.
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Effective Strategies to Overcome the Insolubility of Recombinant ScFv Antibody against EpCAM Extracellular Domain in E. coli Reyhaneh Najafi Soulari1 · Majid Basafa2 · Masoumeh Rajabibazl3,4 · Atieh Hashemi2 Accepted: 31 January 2020 © Springer Nature B.V. 2020
Abstract The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein overexpressed in epithelial-derived neoplasms and therefore is a highly interesting target for antibody therapy in a wide range of carcinomas. Single chain variable fragment (ScFv) antibodies, generated by the association of the variable heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker, retain the binding properties of classical antibodies. Due to its characteristics, Escherichia coli (E. coli) has inspired a great deal of interest for production of antibody fragments with high concentrations. Here, ScFv against EpCAM extracellular domain (EpEX) was expressed in E. coli BL21™(DE3) strain. The effect of different expression conditions on the total protein level was also investigated. Moreover, an attempt was made to overcome the problem of insolubility of the recombinant protein with alterations of expression condition like inducer concentration and temperature as well as addition of the solubility-enhancing agents. Our results showed that the maximum total protein expression was attained 7 h after induction at 37 °C with 0.5 mM IPTG (663.53 ± 7.33 mg/l). Moreover, the expressed antiEpEX-ScFv protein was 39.8% or 29.1% soluble in the presence of 50% glycerol, and Tween20 plus 50% glycerol respectively. Although the solubility of recombinant protein was significantly increased from 39.8% at 37 °C to 43.7% at 16 °C, the maximum level of soluble recombinant protein was attained at 37 °C. Consequently, we report a strategy combining different culture conditions and the solubility-enhancing additives such as glycerol for improving protein solubility. Keywords Escherichia coli · Expression · EpCAM · ScFv · AntiEpEX · Solubility
Introduction
Reyhaneh Najafi Soulari and Majid Basafa have contributed equally in this study. * Atieh Hashemi [email protected]; [email protected] 1
Department of Biotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
2
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, No. 2660, Vali‑e‑Asr Ave., Tehran 1991953381, Iran
3
Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4
Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
The epithelial cell adhesion molecule (EpCAM) is a type-I transmembrane protein involved in cell signaling, proliferation, differentiation, and migration via Ca2 + -independent homotypic cell–cell adhesion activity (Martowicz et al. 2016). EpCAM is expressed only on the basolateral c
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