Naoto Nemoto

In vitro virus: Bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro

Adequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of ‘in vitro virus’ was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell‐free translation system. Bonding efficiency was ∼10%, thus indicating a population of the in vitro virus to have ∼1012 protein variants, this number being 104 that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions.

FLUORESCENCE LABELING OF THE C-TERMINUS OF PROTEINS WITH A PUROMYCIN ANALOGUE IN CELL-FREE TRANSLATION SYSTEMS

We have developed a new method for the C‐terminus‐specific fluorescence labeling of proteins. This method is based on the experimental finding that a fluorescent puromycin analogue at lower concentrations bonds efficiently to the C‐terminus of mature proteins in cell‐free translation systems using mRNA without a stop codon. This labeling is performed under moderate conditions and its labeling efficiency is in the range of 50–95%. Here we demonstrate a protein‐protein interaction assay using fluorescence polarization measurement. This labeling method should also be useful for other rapid molecular interaction assays without purification of the labeled proteins, such as fluorescence correlation spectroscopy.