In vitroselection and evolution of functional proteins by using ribosome display

• DOI: 10.1073/pnas.94.10.4937
• Publication Date: 2002-07-26T14:31:39Z
• Authors: Jozef Hanes; Andreas Plückthun
• Keywords: 0301 basic medicine; Protein Folding; Binding Sites; Base Sequence; Transcription, Genetic; Molecular Sequence Data; Protein Engineering; Biological Evolution; Polymerase Chain Reaction; Recombinant Proteins; 03 medical and health sciences; Protein Biosynthesis; Mutagenesis, Site-Directed; Point Mutation; Amino Acid Sequence; RNA, Messenger; Immunoglobulin Fragments; Ribosomes; DNA Primers; Information Systems

We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 108-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutatedin vitroby acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.