antibody maturation

Alanine scanning mutagenesis is our favorite method in affinity maturation of peptide binders. In this method, each single AA of a selected binding peptide will be replaced with an alanine, and then the binding of the modified peptides to the target protein will be assayed using Biacore technology. The non-essential AAs will be specifically identified. After that, we will create a directed/constrained peptide sub-library that contains random sequences on the non-essential AA positions. Here, again, we frequently randomize the non-essential residues using "NNK" or "trimer codon" strategy. Mutants with increased binding affinity are identified by enhancing the screening stringency, followed by phage ELISA.