mRNA-display-based selections for proteins with desired functions: A protease-substrate case study

Abstract

mRNA-display is an amplification-based, iterative rounds of in vitro protein selection technique that circumvents a number of difficulties associated with yeast two-hybrid and phage display. Because of the covalent linkage between the genotype and the phenotype, mRNA-display provides a powerful means for reading and amplifying a peptide or protein sequence after it has been selected from a library with a diversity in the range of 1012-10 13. In this paper, we briefly review the recent progress in using mRNA-display to identify affinity reagents, binding partners, and enzyme substrates from synthetic peptide or natural proteome libraries. To facilitate the use of mRNA-display in research laboratories without previous experience, we provide a detailed analysis of the critical steps of an mRNA-display-based selection in a case study for the identification of the natural substrates of caspases, including the generation of an mRNA-displayed proteome library, removal of abundant sequences, and selection of proteins with desired functions. The advantages and technical limitations of mRNA-display as a general peptide or protein selection tool are also addressed. © 2008 American Chemical Society and American Institute of Chemical Engineers.