title: Construction and characterization of affibody-Fc chimeras produced in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Biotechnology, Royal Institute of Technology/SCFAB, SE-106 91 Stockholm, Sweden.
authors.forename: Jenny
authors.initials: J
authors.lastname: Rönnmark
authors.forename: Marianne
authors.initials: M
authors.lastname: Hansson
authors.forename: Thien
authors.initials: T
authors.lastname: Nguyen
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Alain
authors.initials: A
authors.lastname: Robert
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.forename: Per-Ake
authors.initials: PA
authors.lastname: Nygren
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001483:Base Sequence; D015374:Biosensing Techniques; D015153:Blotting, Western; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015707:HN Protein / Q000276:immunology; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D019914:Taq Polymerase / Q000276:immunology; D014759:Viral Envelope Proteins
chemical_list: D004274:DNA, Recombinant; D015707:HN Protein; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C116204:attachment protein G; D019914:Taq Polymerase