ENGINEERING THE STEROID-SPECIFICITY OF AN ANTI-17-BETA-ESTRADIOL FAB BY RANDOM MUTAGENESIS AND COMPETITIVE PHAGE PANNING

We have employed random mutagenesis and phage display to improve the s teroid-specificity of an anti-17 beta-estradiol Fab fragment. The V-H domain was mutated using error-prone PCR; the mutation rate was contro lled by adjusting the number of effective duplications. A phage librar y of 2 x 10(6) independent mutants was generated, each mutant containi ng on average 2-4 amino acid changes. We selected for decreased testos terone (TES) cross-reactivity by adding a large excess TES as a compet itor to the panning reactions. After four panning rounds, the cross-re activities of the individual mutant clones ranged from 19 to 4%, showi ng up to 20-fold improvement over the original value (78%), Estradiol affinities were mainly unchanged. Sequencing of the V-H regions reveal ed two hot spots, one located around Ser32 in CDR1 and the other aroun d Thr52(A) in CDR2, while no mutations were found in CDR3., Although m ost clones had multiple mutations, it was possible to deduce the resid ues relevant to the improved specificity by comparing the sequences an d binding data of the mutants. We demonstrated that controlled error-p rone PCR mutagenesis is a rapid method to identify such key residues, lending itself to the scanning of 'lead' positions for further mutagen esis by other methods.