Evolution of antibodies in vitro by ribosome display.
Ribosome display is a cell-free technology which enables in vitro selection and evolution of antibodies from very large diversified DNA libraries. It operates through the following key steps: (1) generation of PCR library; (2) formation of stable antibody-ribosome-mRNA (ARM) complexes as the selection particles, (3) selection of ligand-binding ARM complexes on an immobilized ligand and (4) recovery of the selected genetic information as DNA by RT-PCR. Since PCR-based random or/and site-directed mutagenesis can be easily used to introduce mutations into the selected DNA pool in each cycle, ribosome display offers an efficient "protein evolution" tool for antibody optimization. Both prokaryotic and eukaryotic cell-free systems have been explored for ribosome display of different proteins. In this chapter, we describe the use of the eukaryotic rabbit reticulocyte ribosome display method to isolate variants of V(H) antibody fragments with improved affinities.
Crescendo Biologics Ltd., Babraham Research Campus, Cambridge, UK.
Bryan M
BM
Edwards
Mingyue
M
He
D000818:Animals; D000906:Antibodies / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D055767:Immobilized Proteins / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism*; D013698:Templates, Genetic
D000906:Antibodies; D055767:Immobilized Proteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D004247:DNA