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.
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This study was funded by the research deputy of Shahid Beheshti University of Medical Sciences in Tehran, Iran.
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Atieh Hashemi and Masoumeh Rajabibazl contributed to the study conception and design. Material preparation, data collection and analysis were performed by Reyhaneh Najafi Soulari, Majid Basafa and Atieh Hashemi. The first draft of the manuscript was written by Atieh Hashemi and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Soulari, R.N., Basafa, M., Rajabibazl, M. et al. Effective Strategies to Overcome the Insolubility of Recombinant ScFv Antibody against EpCAM Extracellular Domain in E. coli. Int J Pept Res Ther 26, 2465–2474 (2020). https://doi.org/10.1007/s10989-020-10044-4
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DOI: https://doi.org/10.1007/s10989-020-10044-4