Protein selection using mRNA display

Abstract

mRNA display is an in vitro technique that may be used to search natural or synthetic DNA libraries for the functional proteins and peptides they encode. mRNA-displayed proteins are constructs in which a protein is covalently attached to the RNA that encodes it. This direct covalent association of phenotype (protein) and genotype (RNA) renders the protein directly amplifiable. This in turn allows successive cycles of selection, enrichment, and, optionally, mutagenesis, to be performed upon libraries of displayed proteins. At the end of this process, functional sequences will dominate the library; cloning and sequencing will reveal the identity of the selected functional proteins. mRNA display allows new functional proteins to be discovered without resorting to protein design. This unit describes generation of mRNA-displayed proteins by the in vitro translation of mRNA display templates which are mRNA molecules 3'-terminated in puromycin. Puromycin is a translation inhibitor that is able to enter the ribosome during translation and form a stable covalent bond with the nascent protein. This allows a stable covalent linkage to be formed between the mRNA display template and the protein it encodes, resulting in an mRNA-displayed protein.