Efficiency of puromycin-based technologies mediated by release factors and a ribosome recycling factor

摘要

The identification of protein-protein interaction (PPI) networks is an important aspect of proteomics research. In vitro selection experiments using mRNA display methods, which were originally developed for evolutionary protein engineering, such as in vitro virus (IW) (Nemoto et al, 1997; Miyamoto-Sato et al, 2000; Miyamoto-Sato et al, 2003) or mRNA-peptide fusions (Roberts and Szostak, 1997; Hammond et al, 2001; Keefe and Szostak, 2001), are powerful tools for the analysis of protein function (Amstutz et al, 2001, Miyamoto-Sato et al, 2010). During the course of IW development (Nemoto et al, 1997), two useful puromycin-based techniques (known as 'puromycin TTnive.rsitv PrpQQ technology', Fig. 1A and B) were developed based on the observation that puromycin can bind the C-terminus of a full-length protein (Miyamoto-Sato et al, 2000). One of these puromycin-based techniques is IW formation (Nemoto et al, 1997), whereby an in v/fra-translated full-length protein (phenotype) is attached to its encoding mRNA (genotype). The second puromycin-based technique is C-terminal protein labelling {Nemoto et al, 1999; Miyamoto-Sato et al, 2000; Doi et al, 2002), in which a puromycin derivative bearing a fluorescent moiety is used to label the C-terminal end of a full-length protein. Thus, high-throughput in vitro protein selection may be achieved by combining IW and post-selection analysis (to confirm PPIs) using C-terminally labelled proteins, particularly during proteome exploration.