Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling

The possibility of increasing the affinity of a Tag DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A, A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Tag DNA polymerase binding surface, After selections using monovalent phage display technology, second generation variants were identified having affinities (K-D) for Tag DNA polymerase in the range of 30-50 nM as determined by biosensor technology, Analysis of binding data indicated that the increases in affinity were predominantly due to:decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Tag DNA. polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Tag DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.