Abstract
In the previous study, we constructed an expression vector carrying the anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with VL-linker-VH 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 pelB 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.
Highlights
Antibodies have crystallizable fragment (Fc) domain which binds to Fc receptors and recruits cytotoxic effector molecules and, by interacting with the neonatal Fc receptor, provides long serum half-lives [1, 2]
Construction and Periplasmic Expression of the Anti-EGFRvIII ScFv Antibody Gene in E. coli 143 of the tumour-binding antibody B72.3 with the orientation variable light (VL)-linker-variable heavy (VH) showed greater binding activity than those with the orientation VH-linker-VL
The expression vector was prepared by digestion of pJ414-single-chain Fv (scFv) using NcoI and ClaI restriction enzymes (Fig. 1A)
Summary
Antibodies have crystallizable fragment (Fc) domain which binds to Fc receptors and recruits cytotoxic effector molecules and, by interacting with the neonatal Fc receptor, provides long serum half-lives [1, 2]. Construction and Periplasmic Expression of the Anti-EGFRvIII ScFv Antibody Gene in E. coli 143 of the tumour-binding antibody B72.3 with the orientation VL-linker-VH showed greater binding activity than those with the orientation VH-linker-VL. Our previous study has successfully constructed and expressed an anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation in E. coli periplasm [16].
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