Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.
The possibility of increasing the affinity of a Taq 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 Taq DNA polymerase binding surface. After selections using monovalent phage display technology, second generation variants were identified having affinities (K(D)) for Taq 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 Taq 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 Taq 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.
Gunneriusson
E
E
Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm, Sweden.
Nord
K
K
Uhlén
M
M
Nygren
P
P
D000906:Antibodies / Q000235:genetics* / Q000378:metabolism*; D000918:Antibody Specificity / Q000235:genetics*; D015374:Biosensing Techniques; D019281:Dimerization; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D017433:Protein Structure, Secondary / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019914:Taq Polymerase / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D007074:Immunoglobulin G; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D019914:Taq Polymerase