Engineering the steroid-specificity of an anti-17beta-estradiol Fab by random mutagenesis and competitive phage panning

We have employed random mutagenesis and phage display to improve the steroid-specificity of an anti-17beta-estradiol Fab fragment. The VH domain was mutated using error-prone PCR; the mutation rate was controlled by adjusting the number of effective duplications. A phage library of 2 x 10(6) independent mutants was generated, each mutant containing on average 24 amino acid changes. We selected for decreased testosterone (TES) cross-reactivity by adding a large excess TES as a competitor to the panning reactions. After four panning rounds, the cross-reactivities of the individual mutant clones ranged from 19 to 4%, showing up to 20-fold improvement over the original value (78%). Estradiol affinities were mainly unchanged. Sequencing of the VH regions revealed two hot spots, one located around Ser32 in CDR1 and the other around Thr52A in CDR2, while no mutations were found in CDR3. Although most clones had multiple mutations, it was possible to deduce the residues relevant to the improved specificity by comparing the sequences and binding data of the mutants. We demonstrated that controlled error-prone PCR mutagenesis is a rapid method to identify such key residues, lending itself to the scanning of 'lead' positions for further mutagenesis by other methods.