Construction and characterization of affibody-Fc chimeras produced in Escherichia coli.

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.

Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1M and taq DNA polymerase.

Here we show that robust and small protein ligands can be used for affinity capture of recombinant proteins from crude cell lysates. Two ligands selectively binding to bacterial Taq DNA polymerase and human apolipoprotein A-1(M), respectively, were used in the study. The ligands were selected from libraries of a randomized alpha-helical bacterial receptor domain derived from staphylococcal protein A and have dissociation constants in the micromolar range, which is typical after primary selection from these libraries consisting of approximately 40 million different members each. Using these ligands in affinity chromatography, both target proteins were efficiently recovered from crude cell lysates with high selectivities. No loss of column capacity or selectivity was observed for repeated cycles of sample loading, washing and low pH elution. Interestingly, column sanitation could be performed using 0. 5 M sodium hydroxide without significant loss of ligand performance. The results suggest that combinatorial approaches using robust protein domains as scaffolds can be a general tool in the process of designing purification strategies for biomolecules.

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.

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: the Fab is directed against an intermediate in the helix-coil dynamics of the enzyme.

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a gamma turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.

Evaluation of PCR assays in presence of antibody to thermostable DNA polymerases for detection of microbial agents: avoiding false negative results for specimen containing low-titer agent.

The serial low-titer specimens of Influenza A virus and Adeno virus type 7 were tested for the presence of virus specific genes by PCR based on Tth DNA polymerase and by that based on Taq DNA polymerase, in the absence and presence of antibody to the respective DNA polymerases. Increased product DNA synthesis and higher sensitivity of detection were observed in the presence of antibody compared to those in the absence of antibody. 10- to 100- fold lower titer specimen of Influenza A virus and 10-fold lower titer specimen of Adeno virus could be detected in the presence of antibody than those detected in the absence of antibody to the appropriate DNA polymerase, in a PCR.