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Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1
Author(s) -
Nuttall Stewart D.,
Humberstone Karen S.,
Krishnan Usha V.,
Carmichael Jennifer A.,
Doughty Larissa,
Hattarki Meghan,
Coley Andrew M.,
Casey Joanne L.,
Anders Robin F.,
Foley Michael,
Irving Robert A.,
Hudson Peter J.
Publication year - 2004
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20005
Subject(s) - plasmodium falciparum , biology , selection (genetic algorithm) , computational biology , genetics , malaria , evolutionary biology , computer science , immunology , artificial intelligence
Abstract 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 = ∼2 × 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 ∼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. Proteins 2004;00:000–000. © 2004 Wiley‐Liss, Inc.