Cloning, functional expression and kinetic characterization of pesticide-selective F_ab fragment variants derived by molecular evolution of variable antibody genes

F_ab antibody fragments were constructed by subcloning single chain Fv variable regions from the phagemid vector pCANTAB 5E into the expression vector pASK99. The vector was designed for bacterial secretion of F_ab fragments and bears coding sequences for murine constant domains including the Strep-tag II at the carboxylterminal end of the constant heavy chain domain. The cloning procedure was carried out with the scFv antibodies IPR-7, IPR-53 and IPR-23. The second and third clone originated from the molecular evolution of the s-triazine selective antibody IPR-7. The F_ab fragments were expressed under the transcriptional control of the tetA promoter system. Large-scale production benefits from the anhydrotetracycline-inducible system because of the lower costs for the inducer compared to IPTG and the tightly regulated expression of the recombinant antibody fragments. The Strep-tag purification technology facilitates the isolation of F_ab fragments from the E. coli periplasm. The characteristics of functionally expressed F_ab fragments were determined by employing a BIAcore 2000 system. The K_D of the F_ab variant IPR-23 (K_D=1.12×10^-9 M) optimized by molecular evolution was improved by a factor of 24 compared to the F_ab IPR-7 (K_D=2.73×10^-8 M), which was derived from the template scFv antibody IPR-7. The affinity alteration was also reflected in the 22-fold reduction of the IC₅₀ values of the variants F_ab IPR-7 (IC₅₀=60.5 μg/L) and F_ab IPR-23 (IC₅₀=2.7 μg/L) in the corresponding atrazine ELISA.