Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer...

We have previously generated an affibody molecule for the disease-associated amyloid beta (Aβ) peptide, which has been shown to inhibit the formation of various Aβ aggregates and revert the neurotoxicity of Aβ in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the Aβ-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and Aβ binding. Overall, the detailed engineering and characterization of this promising Aβ-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins.