The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains. The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications.
The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysinespecific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (K d = 1.31 þ 0.26U U10 37 M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody-like molecule. ß
The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (V NAR ), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (V NAR 12F-11) after four rounds of in vitro selection. V NAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mgAEL ) indicating that these V NAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing ÔintrabodyÕ variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular V NAR is critically dependent upon precise CDR loop conformations for its binding affinity.Keywords: new antigen receptor; variable domain; peptide display; Tom70; mitochondrial import.Conventional antibodies recognize antigens through the combination of six complementarity determining region (CDR) loops displayed three each upon variable heavy (V H ) and variable light (V L ) chain immunoglobulin domains [1]. These CDR loops vary in size and composition allowing formation of a large number of conformational antigenbinding surfaces including planar and ridged topologies [2]. The orientation of the loops is maintained by a combination of their internal architecture, the underlying immunoglobulin scaffold, and the hydrophobic interaction between the antibody V H and V L domains [3]. In contrast, families of antibody-like molecules characterized recently in camelids and sharks rely on a single immunoglobulin V H -like domain framework, which presents two or three CDR loops to form the antigen-binding interface [4][5][6][7]. For camelids, these V H H single domain antibodies can bind an extensive range of antigens, including large proteins, enzymes (either within or outside the active site clefts), haptens and dyes. Biochemical and structural data now shows that V H H binding affinity resides in a variety of possible CDR conformations that can include all three CDRs, or an elongated CDR3 loop alone, or a combination of CDR and framework side-chain and main-chain residues [8].For sharks, the new antigen receptor (IgNAR) from Ginglymostoma cirratum (nurse sharks) and Orectolobus maculatus (wobbegong sharks) also utilizes a single V H -like domain, which we herein term V NAR [9,10]. Structurally, the entire intact IgNAR a...
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