tailieunhanh - Báo cáo khoa học: 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 disul-phide 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 con-structed anin 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 (VNAR12F-11) after four rounds ofin vitroselection | Eur. J. Biochem. 270 3543-3554 2003 FEBS 2003 doi Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70 Stewart D. Nuttall1 2 Usha V. Krishnan1 2 Larissa Doughty1 Kylie Pearson2 3 Michael T. Ryan2 3 Nicholas J. Hoogenraad2 3 Meghan Hattarki1 Jennifer A. Carmichael1 2 Robert A. Irving1 2 and Peter J. Hudson1 2 1CSIRO Health Sciences and Nutrition and 2CRC for Diagnostics Parkville Victoria Australia 3Department of Biochemistry La Trobe University Bundoora Victoria Australia 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 I unciions 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 L-1. Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected b-sheet confor mation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore Kd 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 die impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High .

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