title: Antibody binding loop insertions as diversity elements.
abstract: In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.
authors.affiliation: HCDR3s as diversity elements, Los Alamos National Laboratory, Los Alamos, NM, USA.
authors.forename: Csaba
authors.initials: C
authors.lastname: Kiss
authors.forename: Hugh
authors.initials: H
authors.lastname: Fisher
authors.forename: Emanuele
authors.initials: E
authors.lastname: Pesavento
authors.forename: Minghua
authors.initials: M
authors.lastname: Dai
authors.forename: Rosa
authors.initials: R
authors.lastname: Valero
authors.forename: Milan
authors.initials: M
authors.lastname: Ovecka
authors.forename: Rhiannon
authors.initials: R
authors.lastname: Nolan
authors.forename: M Lisa
authors.initials: ML
authors.lastname: Phipps
authors.forename: Nileena
authors.initials: N
authors.lastname: Velappan
authors.forename: Leslie
authors.initials: L
authors.lastname: Chasteen
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Martinez
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Waldo
authors.forename: Peter
authors.initials: P
authors.lastname: Pavlik
authors.forename: Andrew R M
authors.initials: AR
authors.lastname: Bradbury
mesh_terms: D000916:Antibody Diversity; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D022801:Complementarity Determining Regions / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D017422:Sequence Analysis, DNA
chemical_list: D022801:Complementarity Determining Regions; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009113:Muramidase