In vitro selection and evolution of functional proteins by using ribosome display.
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 10(8)-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by 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.
Hanes
J
J
Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Plückthun
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D017931:DNA Primers; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry*; D007256:Information Systems; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D014158:Transcription, Genetic
D017931:DNA Primers; D007128:Immunoglobulin Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins