Modified peptide selection in vitro by introduction of a protein-RNA interaction.
The ribosome display system is a very effective and powerful tool for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. The system depends on the stability of ribosome-mRNA complexes that have been formed as a result of the removal of a stop codon. To assess the general applicability of the system, we examined the stability of ribosome-mRNA complexes in the presence and absence of a stop codon, as well as in the presence and the absence of an additional interaction between the translated peptide and its mRNA within the ribosome-mRNA complex. The additional interaction that we exploited was the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (Cv). The MSp dimer and Cv were placed, respectively, at the N-terminal end of a nascent protein, translated in vitro, and at the 5' end of the protein's mRNA, and consequently further stabilize the ribosome-mRNA complex. To our surprise, we were able to select proteins even in the presence of a stop codon. Moreover, as we had anticipated, the interaction between the MSp dimer and Cv enhanced the stability of the ribosome-mRNA complex, suggesting that this kind of interaction might be useful in the design of an efficient ribosome display selection strategy. Indeed, the yield of the mRNAs of interest after selection was increased upon the introduction of the interaction between the MSp dimer and Cv.
Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan.
Shinya Y
SY
Sawata
Kazunari
K
Taira
D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D018388:Codon, Terminator; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D036022:Capsid Proteins; D018388:Codon, Terminator; D010455:Peptides; D016601:RNA-Binding Proteins; D012313:RNA