Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.
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 (VNAR), 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 (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR 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 'intrabody' 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 VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.
Nuttall
Stewart D
SD
CSIRO Health Sciences and Nutrition, Parkville, Victoria, Australia. Stewart.Nuttall@csiro.au
Krishnan
Usha V
UV
Doughty
Larissa
L
Pearson
Kylie
K
Ryan
Michael T
MT
Hoogenraad
Nicholas J
NJ
Hattarki
Meghan
M
Carmichael
Jennifer A
JA
Irving
Robert A
RA
Hudson
Peter J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D002942:Circular Dichroism; D022801:Complementarity Determining Regions / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007136:Immunoglobulins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011946:Receptors, Antigen / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012754:Sharks / Q000276:immunology*
D022801:Complementarity Determining Regions; D017931:DNA Primers; D004220:Disulfides; D005656:Fungal Proteins; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D008565:Membrane Proteins; D019151:Peptide Library; D011946:Receptors, Antigen; D011994:Recombinant Proteins; C092388:nurse shark antigen receptor