Ribosome display of antibodies: expression, specificity and recovery in a eukaryotic system.
In ribosome display, proteins are linked to their encoding genetic material as protein-ribosome-mRNA complexes. The technology has been applied to the isolation of antibodies and other proteins from large PCR-derived libraries. Here we demonstrate the specificity of eukaryotic ribosome complexes and investigate recovery and display procedures using a single chain version of the anti-progesterone monoclonal antibody DB3. Complexes are formed by deletion of the 3' stop codon in a coupled rabbit reticulocyte system. Using inhibition with different steroid probes, we show that the fine specificity of the combining site expressed as a nascent protein is closely similar to the native monoclonal, indicating correct folding and function while bound to the ribosome. We have demonstrated that the 3' end of the mRNA is blocked by the stalled ribosome and unavailable to primers. Moreover, we show that an in situ RT-PCR recovery procedure, carried out on intact complexes, is more efficient than ribosome disruption and isolation of mRNA followed by RT-PCR. We also explore the Mg(2+) and DTT concentrations and time required for efficient production of complexes. Our findings confirm the effectiveness of the eukaryotic ribosome display system and define conditions for efficient selection of single chain antibodies.
He
Mingyue
M
Protein Technologies Laboratory, The Babraham Institute, The Babraham Research Campus, Cambridge CB2 4AT, UK.
Taussig
Michael J
MJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D002474:Cell-Free System; D018388:Codon, Terminator / Q000235:genetics; D019151:Peptide Library; D011374:Progesterone / Q000276:immunology; D014176:Protein Biosynthesis / Q000187:drug effects / Q000502:physiology; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D013256:Steroids / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D018388:Codon, Terminator; D019151:Peptide Library; D012333:RNA, Messenger; D013256:Steroids; D011374:Progesterone