Selection of proteins with desired properties from natural proteome libraries using mRNA display

Abstract

mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca(2+)-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display-based selection can be accomplished in ~7 d.

LOVTRAP: an optogenetic system for photoinduced protein dissociation

“…21 Reverse transcription was performed to convert the mRNA-protein into cDNA/mRNA-protein form. The purified fusion library was diluted in a binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20, 1 mg/mL BSA, 1 mg/mL yeast tRNA and 0.5 mM EDTA) and passed through 75 μL Streptavidin UltraLink Plus Resin (Thermo Scientific) pretreated with 1 mg/mL BSA and 1 mg/mL yeast tRNA to minimize the sequences that nonspecifically bound to streptavidin and/or the matrix.…”

Peptide Aptamers: Development and Applications

“…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. Some problems are intrinsic to all extracellular display methods that employ multiple amplification/selection rounds.…”

Development of Next-Generation Peptide Binders Using In vitro Display Technologies and Their Potential Applications

“…Against the above backdrop, ribosome display, mRNA display, and CIS display have been developed as in vitro , cell free, display technologies in combination with cell-free protein synthesis systems (24–26). In selecting peptide binders using these technologies, the preparation and use of transition complexes that link combinatorial peptides (phenotype) and the corresponding mRNAs or DNAs (genotype) is indispensable.…”

“…However, to link combinatorial peptides (phenotype) and their mRNAs (genotype), covalently linked peptide–mRNA complexes are used for selection (25). The covalent linkage in the complex is synthesized via reaction of the C-terminus of a nascent polypeptide with puromycin in the A-site of the ribosome through the formation of the corresponding peptide–ribosome–mRNA–puromycin complex.…”