Puromycin technology for in vitro evolution and proteome exploration

Abstract

  Puromycin, an analogue of the 3’ end of aminoacyl-tRNA, is transferred non-specifically to growing polypeptide chains, causing premature termination of translation. However, we found that, at very low concentration, puromycin is transferred specifically to the carboxyl (C-) terminus of the full-length protein. The term puromycin technology refers to methods which have been developed based on this property, including the in vitro virus (IVV) display technique, in which an mRNA (genotype) and its protein (phenotype) are linked via puromycin, and the technique of specific in vitro C-terminal protein labeling. Novel and convenient applications based on puromycin technology have been employed not only for evolutionary protein engineering, such as protein-directed selection, but also in various fields of proteomics research, such as fluorescence labeling, affinity purification (pull-down assay), protein-protein interaction analysis, and protein chips. Here, we review the properties of puromycin that make this technology possible, and the already extensive range of applications of this technology.