Generation of a phagemid mouse recombinant antibody fragment library by multisite-directed mutagenesis.
A nonimmune phagemid recombinant antibody fragment (rFab) library was generated with a nominal diversity of 1.16 x 10(7) using the QuikChange Multi Site-Directed Mutagenesis kit. Two degenerate primers spanning the third complementarity-determining region (CDR) loops of the antibody fragment light and heavy chain were mutated such that eight or nine amino acids were randomly changed per CDR loop. Seven proteins were used to evaluate the library quality. Protein-specific rFab antibodies were selected after three panning cycles. From 12% to 64% of the randomly selected colonies produced positive ELISA signals to the phagemid rFabs. Multisite-directed mutagenesis allowed a diverse rFab library to be rapidly constructed while retaining the structural framework of a Fab that had been optimized for production in Escherichia coli.
University of Georgia, Athens, GA, USA.
Laura-Lee Clancy
LL
Kelley
Cory
C
Momany
D000818:Animals; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000096:biosynthesis
D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins