Antibody binding loop insertions as diversity elements

Csaba Kiss, Hugh Fisher, Emanuele Pesavento, Minghua Dai, Rosa Valero, Milan Ovecka, Rhiannon Nolan, M. Lisa Phipps, Nileena Velappan, Leslie Chasteen, Jennifer S. Martinez, Geoffrey S. Waldo, Peter Pavlik, Andrew R.M. Bradbury

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

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.

Original languageEnglish (US)
Article numbere132
JournalNucleic Acids Research
Volume34
Issue number19
DOIs
StatePublished - Nov 2006
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

Fingerprint

Dive into the research topics of 'Antibody binding loop insertions as diversity elements'. Together they form a unique fingerprint.

Cite this