A novel MRGPRX2-targeting antagonistic DNA aptamer inhibits histamine release and prevents mast cell-mediated anaphylaxis.

Anaphylaxis during general anaesthesia is a significant clinical challenge for anaesthesiologists. Approximately 50% of perioperative anaphylaxis cases lack the presence of specific IgE antibodies. Mas-related G-protein coupled receptor X2 (MRGPRX2) in humans and its mouse orthologue Mas-related G-protein coupled receptor B2 (Mrgprb2) are crucial receptors in non-IgE-dependent histamine release.

Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.

G-protein-coupled receptors (GPCRs) are membrane proteins distributed on the cell surface, and they may be potential drug targets. However, synthesizing GPCRs can be challenging. Recently, some cell-free protein synthesis systems have been shown to produce a large amount of membrane protein combined with chemical chaperones that include liposomes and glycerol.

Efficiency and Safety of CRAC Inhibitors in Human Rheumatoid Arthritis Xenograft Models.

Store-operated Ca release-activated Ca (CRAC) channels are involved in the pathogenesis of rheumatoid arthritis (RA) and have been studied as therapeutic targets in the management of RA. We investigated the efficacy and safety of CRAC inhibitors, including a neutralizing Ab (hCRACM1-IgG) and YM-58483, in the treatment of RA. Patient-derived T cell and B cell activity was suppressed by hCRACM1-IgG as well as YM-58483.

Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology.

The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes.

G-protein coupled receptors (GPCRs) share a common seven-transmembrane topology and mediate cellular responses to a variety of extracellular signals. However, structural and functional approaches to GPCRs have often been limited by the difficulty of producing a sufficient amount of receptor protein using conventional expression systems. We synthesized human dopamine D1 receptors using a wheat cell-free protein synthesis system with liposomes and then analyzed their receptor binding ability.

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

Background: Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system.

Purification of eukaryotic translation factors from wheat germ for reconstitution of protein synthesis.

The wheat germ cell-free protein synthesis is a powerful and versatile method for preparation of proteins based on the accumulated DNA sequence information. As the cell extract used for it contains many factors that are unknown or do not directly involve in protein synthesis, details of the translation reaction is yet to be understood. Therefore, we have decided to try reconstitution of protein synthesis, which would be useful for better understanding of the mechanisms supporting eukaryotic protein synthesis and translational regulation and probably applicable to synthetic biology.

Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.

A cell-free protein synthesis system is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. Here, the authors describe unnatural amino acid probing in a wheat germ cell-free translation system as a method for detecting the structural changes that occur in a cofactor binding protein on a conversion of the protein from an apo-form to a holo-form. The authors selected the FMN-binding protein from Desulfovibrio vulgaris as a model protein.

Application of cell-free translation systems to studies of cofactor binding proteins.

To develop applications of in vitro cell-free translation systems for production and characterization of cofactor binding proteins, we investigate the production of apo- or holo-forms of Flavin Mono Nucleotide (FMN)-binding protein from Desulfovibrio vulgaris (Miyazaki F) and purified them. The redox potential analysis and measurements of UV-, visible, and fluorescent spectra of reconstructed holo-protein showed that the FMN correctly bound to the FMN binding site. On the other hand, contrary to our expectation, we found that the apo-protein formed a dimer structure and the incorporation of the FMN led the conformational alterations of the protein.

A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system.

For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms.

Formation of circular polyribosomes in wheat germ cell-free protein synthesis system.

We report a morphological study of functioning ribosomes in a efficient and robust cell-free protein synthesis system prepared from wheat embryos. Sucrose density gradient analysis of translated mixtures programmed with luciferase mRNAs having different 5' and 3' untranslated regions showed formation of large polysomes. Electron microscopic examination of translation mixtures programmed with those of capped and polyadenylated mRNA revealed that ribosomes assemble into a circular-type polysome in vitro.

Aquifex aeolicus tRNA (Gm18) methyltransferase has unique substrate specificity. TRNA recognition mechanism of the enzyme.

Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme.

A cell-free protein synthesis system for high-throughput proteomics.

We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel.

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.