Toshihiro Kuroita

A bilayer cell-free protein synthesis system for high-throughput screening of gene products

A high‐throughput cell‐free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch‐mode reaction. Combining this method with a wheat germ cell‐free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer‐based cell‐free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post‐genome era.

Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification

Magnetic particles are useful for simple and efficient nucleic acid extraction. To achieve fully automated nucleic acid extraction and purification using magnetic particles, a new method for operating magnetic particles, Magtration Technology, was developed. In this method, magnetic separation is performed in a specially designed disposable tip. This enables high recovery of magnetic particles with high reproducibility. The features of this technology are (i) a simple mechanism for process control and (ii) flexible software to enable adaptation to commercially available reagents. Automated instruments based on Magtration Technology were developed and used for nucleic acid extraction. Total DNA, total RNA and plasmids were purified by Magtration Technology at an efficiency comparable to that of manual methods.

Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.

Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique

Specific interactions between transcription factors and cis‐acting DNA sequence motifs are primary events for the transcriptional regulation. Many regulatory elements appear to diverge from the most optimal recognition sequences. To evaluate affinities of a transcription factor to various suboptimal sequences, we have developed a new detection method based on the surface plasmon resonance (SPR) imaging technique. Transcription factor MafG and its recognition sequence MARE (Maf recognition elements) were adopted to evaluate the new method. We modified DNA immobilization procedure on to the gold chip, so that a double‐stranded DNA array was successfully fabricated. We further found that a hydrophilic flexible spacer composed of the poly (ethylene glycol) moiety between DNA and alkanethiol self‐assembled monolayers on the surface is effective for preventing nonspecific adsorption and facilitating specific binding of MafG. Multiple interaction profiles between MafG and six of MARE‐related sequences were observed by the SPR imaging technique. The kinetic values obtained by SPR imaging showed very good correlation with those obtained from electrophoretic gel mobility shift assays, although absolute values were deviated from each other. These results demonstrate that the double‐stranded DNA array fabricated with the modified multistep procedure can be applied for the comprehensive analysis of the transcription factor‐DNA interaction.