Construction and Periplasmic Expression of the Anti-EGFRvIII ScFv Antibody Gene in Escherichia coli

摘要

In the previous study, we constructed an expression vector carrying the anti-EGFRvlll scFv antibody gene with V_H-linker-V_L orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with V_L-linker-V_H orientation of the anti-EGFRvIII scFv gene and analyzed its expression in E. coli. The DNA fragment was amplified from its cloning vector (pTz-rscFv), subsequently cloned into a previous expression vector containing the peIB signal sequence and his-tag, and then transformed into E. coli TOP10. The recombinant plasmids were characterized by restriction, PCR, and DNA sequencing analyses. The new anti-EGFRvIII scFv antibody proteins have been successfully expressed in the periplasmic compartment of E. coli Nico21(DE3) using 0.1 mM final concentration of IPTG induction. Total proteins, soluble periplasmic and cytoplasmic proteins, solubilized inclusion bodies, and extracellular proteins were analyzed by SDS-PAGE and Western Blot analyses. The results showed that soluble scFv proteins were found in all fractions except from the cytoplasmic space.