Affinity maturation of a TNFalpha-binding affibody molecule by Darwinian survival selection

The introduction of different methodologies for the construction and screening of complex protein libraries has provided powerful means in protein engineering for the development of molecules with desired traits. A challenge faced in many situations is to adapt a given methodology for efficient and rapid identification of the most interesting variants present in a library. In the present study, the concept of Darwinian selection based on a growth advantage for clones having the desired trait has been investigated. Using a beta-lactamase-based PCA (protein fragment complementation assay), affinity maturation of a TNFalpha (tumour necrosis factor alpha)-binding Affibody molecule with an initial 2 nM affinity for the target has been performed. Initial characterization of the PCA system, based on the affinity-driven reconstitution of beta-lactamase activity in the periplasm of cells harbouring a library member showing affinity for a co-expressed target protein, showed that the system was responsive to promoter induction level, interaction affinity and applied selection pressure. Using combinatorial protein engineering principles, a 107 library of second-generation Affibody molecules was constructed and subjected to selection of improved variants by library growth in liquid culture. The results show that, after a pre-selection step on semi-solid medium to eliminate non-binding variants, present in the majority, two rounds of selection in liquid culture resulted in an enrichment for binders showing up to 8-fold higher affinity for the TNFalpha target than the ancestral variant. Biosensor analyses showed that the major factor for the improved affinity could be attributed to reduced off-rate constants.