Ribosome display and screening for protein therapeutics.
Ribosome display is a cell-free display technology which enables in vitro selection of antibodies from large recombinant DNA libraries. It also allows continuous introduction of mutations into the selected DNA pool by PCR-based mutagenesis in each cycle, enabling selection of antibody variants with improved affinity, specificity, and stability, thus providing a powerful "protein evolution" tool for optimizing antibody therapeutics. Ribosome display selects required molecules by linking individual proteins (phenotype) with their corresponding mRNAs (genotype) through the formation of stable Protein-Ribosome-mRNA (PRM) complexes. By affinity interaction with an immobilized ligand, the captured PRM complexes are recovered as cDNA using RT-PCR from the ribosome-attached mRNA. The DNA is then subjected to subsequent ribosome display cycles for further enrichment of rare species or cloning, expression, and sequencing to identify wanted candidates. Both prokaryotic and eukaryotic cell-free systems have been developed for ribosome display of different proteins. In this chapter, we describe ribosome display of antibodies using the eukaryotic rabbit reticulocyte system with an in situ single-primer DNA recovery method. A high-throughput Escherichia coli expression format is also described for screening of individual antibody binders from the ribosome-selected population.
University of Ljubljana, Ljubljana, Slovenia.
Damjana
D
Kastelic
Mingyue
M
He
D000818:Animals; D002474:Cell-Free System; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli; D015723:Gene Library; D046911:Macromolecular Substances / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D012156:Reticulocytes; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies / Q000235:genetics / Q000302:isolation & purification*
D018076:DNA, Complementary; D046911:Macromolecular Substances; D011506:Proteins; D012333:RNA, Messenger; D057127:Single-Chain Antibodies