Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems

AbstractWe 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

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

AbstractAdequate 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

Optimization of Ladle Tilting Speed for Preventing Temperature Drops in the Die Casting Process

In die casting, molten metal poured into a shot sleeve is pressed into a mold by a plunger at high speed. The temperature of the metal drops significantly while it is being poured from the ladle to the shot sleeve, resulting in casting defects such as misrun flow lines. Although it is important to control the temperature at all stages of the process, a method for minimizing temperature loss has not yet been clarified to date. In this study, the cause of the temperature drop in the shot sleeve was clarified, and a method of optimizing the ladle tilting speed was proposed to prevent temperature drop. First, experiments were conducted to measure the decrease in metal temperature in the sleeve during pouring. These experiments revealed that the metal cools significantly from the moment it touches the shot sleeve. Therefore, the time from the first contact between the shot sleeve and the metal to the start of pouring was set as the objective function. A genetic algorithm was then used to derive the optimal ladle tilting speed pattern to suppress the temperature drop. This analysis confirmed that the metal was poured without flowing out or running ahead and that the immediate liquid level vibration after pouring was suppressed, thus ensuring stable pouring

Solid-phase translation and RNA–protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA

A novel cell-free translation system is described in which template-mRNA molecules were captured onto solid surfaces to simultaneously synthesize and immobilize proteins in a more native-state form. This technology comprises a novel solid-phase approach to cell-free translation and RNA–protein fusion techniques. A newly constructed biotinylated linker-DNA which enables puromycin-assisted RNA–protein fusion is ligated to the 3′ ends of the mRNA molecules to attach the mRNA-template on a streptavidin-coated surface and further to enable the subsequent reactions of translation and RNA–protein fusion on surface. The protein products are therefore directly immobilized onto solid surfaces and furthermore were discovered to adopt a more native state with proper protein folding and superior biological activity compared with conventional liquid-phase approaches. We further validate this approach via the production of immobilized green fluorescent protein (GFP) on microbeads and by the production and assay of aldehyde reductase (ALR) enzyme with 4-fold or more activity. The approach developed in this study may enable to embrace the concept of the transformation of ‘RNA chip-to-protein chip’ using a solid-phase cell-free translation system and thus to the development of high-throughput microarray platform in the field of functional genomics and in vitro evolution

An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution

The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion (=viral particle)” (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this “viral genome” was demonstrated