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

摘要

F_(ab) antibody fragments were constructed by sub-cloning 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 carboxyl-terminal 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 anhydro-tetracycline-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 IC_(50) values of the variants F_(ab) IPR-7 (IC_(50) = 60.5 μg/L) and F_(ab) IPR-23 (IC_(50) = 2.7 μg/L) in the corresponding atrazine ELISA.