Jun Yokoyama

A practical method for cell-free protein synthesis to avoid stable isotope scrambling and dilution.

During recent years, the targets of protein structure analysis using nuclear magnetic resonance spectroscopy have become larger and more complicated. As a result, a complete and precise stable isotope labeling technique has been desired. A cell-free protein synthesis system is appropriate for this purpose. In the current study, we achieved precise and complete (15)N and (2)H labeling using an Escherichia coli cell extract-based cell-free protein synthesis system by controlling the metabolic reactions in the system with their chemical inhibitors. The addition of aminooxyacetate, d-malate, l-methionine sulfoximine, S-methyl-l-cysteine sulfoximine, 6-diazo-5-oxo-l-norleucine, and 5-diazo-4-oxo-l-norvaline was quite effective for precise amino acid-selective (15)N labeling even for aspartic acid, asparagine, glutamic acid, and glutamine, which generally suffer from severe isotope scrambling and dilution when using the conventional cell-free system. For (2)H labeling, the back-protonation of the H(α) and H(β) positions, which commonly occurred in the conventional system, was dramatically suppressed by simply adding aminooxyacetate and d-malate to the cell-free system except for the H(α) positions in methionine and cysteine.

An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system

Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein using Escherichia coli cell extract-based CF. This method takes advantage of endogenous metabolic conversions to generate SI-labeled asparagine, glutamine, cysteine, and tryptophan, which are much more expensive than the other 16 kinds of SI-labeled amino acids, from inexpensive sources, such as SI-labeled algal amino acid mixture, SI-labeled indole, and sodium sulfide, during the CF reaction. As compared with the conventional method employing 20 kinds of SI-labeled amino acids, highly enriched uniform SI-labeling with similar labeling efficiency was achieved at a greatly reduced cost with the newly developed method. Therefore, our method solves the cost problem of the SI labeling of proteins using the CF.

Rapid biochemical synthesis of 11C-labeled single chain variable fragment antibody for immuno-PET by cell-free protein synthesis

Immuno-PET is a promising approach for improved cancer diagnosis, by taking advantage of the high specificity of antibodies. Here, we present a novel cell-free protein synthesis method for preparing a positron emitter labeled-antibody. Functional anti-human EGFRvIII single chain Fv, MR1-1, was successfully labeled with carbon-11 (half-life=20.4 min) in 5 min (36% yield) by the direct incorporation of the clinical PET tracer, l-[(11)C]methionine. The product [(11)C]MR1-1 was easily and rapidly isolated with high radiochemical purity (>95%) from the reaction solution, by affinity purification. This method would be widely applicable to the preparation of radiolabeled antibodies for PET imaging.

Dielectric Properties in Ferroelectric Liquid Crystal Mixtures with Ultrashort Pitch

The dielectric propereties of smectic liquid crystal mixtures containing chiral dopants derived from trifluoromethylated pyranose were studied. In every mixture, a large dielectric response due to the Goldstone mode was observed, indicating the smectic C* phase, although two switching current peaks indicative of the smectic CA* phase were observed in the mixtures with high dopant ratios. With increasing the chiral dopant ratio, the dielectric strength becomes smaller and the relaxation frequency becomes larger. This behavior was qualitatively explained by the chiral dopant ratio dependence of the spontaneous polarization and the helicoidal pitch. It was also found that a higher dc bias field was required to suppress the Goldstone mode in the mixture with a higher dopant ratio, suggesting the helicoid-stabilized state.

Helicoid-Stabilized Tristable Switching in Ferroelectric Liquid Crystal Mixtures with Ultrashort Pitch

Antiferroelectric-like tristable switching was observed in smectic liquid crystal mixtures containing chiral dopants derived from trifluoromethylated pyranose. The results of transmittance measurement with obliquely incident light clearly indicate that the phase showing the tristable switching is not antiferroelectric but ferroelectric. The helicoidal pitch quadratically changes with the amount of dopant and seems to attain about 50 nm in the mixtures with a dopant content of more than 40 wt%. The quadratic dependence originates from the flexoelectric effect with negligible inter-molecular chiral interaction. Mechanisms for the tristable switching are proposed on the basis of the ultrashort pitch.

Relationship between Flexoelectricity and Helical Pitch in Ferroelectric Liquid Crystal Mixtures Containing Host Achiral Compounds and Chiral Dopants

The helical pitch as a function of the molar ratio of chiral dopants was discussed based on the host-host, host-dopant and dopant-dopant flexoelectric interactions in a ferroelectric liquid crystal mixture system. It was found that the wavenumber of the pitch approximately changes from quadratic to cubic dependence on the chiral dopant ratio and that the host-dopant flexoelectric interactions are negligible compared with the host-host and dopant-dopant interactions.