Abstract
This study examined the global stability and activity properties of recombinant DNA-binding antibody fragments that were obtained from a bacteriophage combinatorial display library. The goal of this study was to determine whether the combinatorial approach of heavy and light chain assembly inE. coliand subsequent affinity selection preferentially selects for antibody fragments with unusual structural stabilities. Specifically, the binding properties and stability of recombinant antibody fragments with or without a C-terminal His tag to temperature, pH, and guanidine–HCl were examined. Both Fab exhibited almost identicalKd(120–130, 140–170, and 450–560 nm) and maximal fluorescence quenching (20–25%) values for binding to (dT)20, (dT)15, and (dT)10, respectively. Thermal denaturation data obtained by CD spectroscopy demonstrated that both Fab possessed structural properties comparable to well-folded proteins with defined tertiary structures which were stable below 70°C (Tm73°C). These results were confirmed by differential scanning calorimetry. Both Fab exhibited the same rate of irreversible thermal inactivation (0.061–0.069 min−1) at 75°C and could be reversibly renatured from guanidine–HCl and pH extremes. Crystallization trials with one recombinant DNA-binding Fab yielded diffraction quality crystals also suggesting a well-defined tertiary structure.
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