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

Abstract

Fab 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 Fab 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 Fab 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 Fab fragments from the E. coli periplasm. The characteristics of functionally expressed Fab fragments were determined by employing a BIAcore 2000 system. The KD of the Fab variant IPR-23 (K(D)= 1.1 2 x 10(-9) M) optimized by molecular evolution was improved by a factor of 24 compared to the Fab IPR-7 (K(D) = 2.73 x 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 Fab IPR-7 (IC50 = 60.5 microg/L) and Fab IPR-23 (IC50=2.7 microg/L) in the corresponding atrazine ELISA.