Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library.
The immunoglobulin isotype IgNAR (Novel Antigen Receptor) was discovered in the serum of the nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates) as a homodimer of two protein chains, each composed of a single variable domain (V) domain and five constant domains. The IgNAR variable domain contains an intact antigen-binding site and functions as an independent domain able to react to antigen with both high specificity and affinity. Here we describe the successful construction of a synthetic phage-displayed library based upon a single anti-lysozyme clone HEL-5A7 scaffold, which was previously selected from an immune IgNAR variable domain library. The complementarity-determining region 3 (CDR3) loop of this clone was varied in both length and composition and the derived library was used to pan against two model proteins, lysozyme and leptin. A single anti-lysozyme clone (Ly-X20) and anti-leptin clone (Lep-12E1) were selected for further study. Both clones were shown to be functionally expressed in Escherichia coli, extremely thermostable and bind to corresponding antigens specifically. The results here demonstrate that a synthetic IgNAR variable domain library based on a single framework scaffold can be used as a route to generate antigen binders quickly, easily and without the need of immunization.
Shao
Cui-Ying
CY
Department of Molecular and Cell Biology, Foresterhill, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK. c.shao@abdn.ac.uk
Secombes
Chris J
CJ
Porter
Andrew J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D007128:Immunoglobulin Fragments / Q000235:genetics; D007132:Immunoglobulin Isotypes / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007136:Immunoglobulins / Q000235:genetics* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011946:Receptors, Antigen / Q000235:genetics* / Q000302:isolation & purification; D016415:Sequence Alignment; D012754:Sharks / Q000276:immunology*
D007128:Immunoglobulin Fragments; D007132:Immunoglobulin Isotypes; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D011946:Receptors, Antigen