In vitro selection of proteins with desired characteristics using mRNA-display

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 very high diversity. The purpose of this article is to provide a summary of the field and practical framework of mRNA-display-based selections. We summarize the advantages and limitations of selections using mRNA-display as well as the recent applications, namely, the identification of novel affinity reagents, target-binding partners, and enzyme substrates from synthetic peptide or natural proteome libraries. Practically, we provide a detailed procedure for performing mRNA-display-based selections with the aim of identifying protease substrates and binding partners of a target protein. Furthermore, we describe how to confirm the function of the selected protein sequences by biochemical assays and bioinformatic tools.

Evolving a Peptide: Library Platforms and Diversification Strategies

“…These include cyclic peptide therapeutics targeting GPCR signaling [12], streptavidin binding peptides [156], and peptide vaccines [157]. Compared to ribosome display, this platform is superior and enables stringent selection conditions [158]. A major advantage over ribosome display is obviously the absence of the ribosome, a huge 2,000,000 Da ribonucleoprotein complex, which can interfere with the selection process [159].…”

“…One of the concerns involves interactions of covalently bound mRNA with the displayed peptide or the target molecule. The interference of flexible mRNA is particularly problematic when dealing with proteins that nonspecifically bind nucleic acids [158]. Furthermore, the highly negatively charged mRNA fusion moiety may interfere with positively charged target molecules [160].…”

Peptide Aptamers: Development and Applications

“…There are several reviews discussing the advantages and limitations of in vitro display methods [166, 170, 181, 196], of which the large library size and independence from cellular physiology are the main benefits. Restrictions in the size of the investigated protein, the inability to deal with protein complexes and membrane components, as well as difficulties of working in an RNAse-free environment [197] are the main limitations.…”

Proteolysis to Identify Protease Substrates: Cleave to Decipher

“…Also, cellular library of peptide substrates (CLiPS) enables the display of fluorescent peptide substrate on the surface of Escherichia coli . The yeast two‐hybrid system and the mRNA display technique have also been reported to be effective in the discovery of protease substrates . Further, proteomics‐based approaches are known to facilitate high‐throughput identification of protease substrates.…”

“…[26] The yeast two-hybrid system and the mRNA display technique have also been reported to be effective in the discovery of protease substrates. [27,28] Further, proteomics-based approaches are known to facilitate high-throughput identification of protease substrates. Gel-based proteomics techniques include 1D and 2D polyacrylamide gel electrophoresis (1D & 2D PAGE) [29] and 2D differential gel electrophoresis (2D DIGE).…”