α-Glucosidases, which catalyze the hydrolysis of α-glucosidic linkage at nonreducing end substrate, are important for metabolism α-glucosides. Halomonas sp. H11 α-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards α-(1 → 4)-linked disaccharide maltose among naturally occurring substrates. Although several three-dimensional structures GH13 members have been solved, specificity and 4) recognition mechanism unclear owing a lack...

The insertion of the DNA sequence encoding SKIK peptide adjacent to M start codon a difficult-to-express protein enhances production in Escherichia coli. In this report, we reveal that increased SKIK-tagged is not due usage sequence. Furthermore, found or MSKIK just before SecM arrest (FSTPVWISQAQGIRAGP), which causes ribosomal stalling on mRNA, greatly containing E. coli-reconstituted cell-free synthesis system (PURE system). A similar translation enhancement phenomenon by was observed for...

Abstract Obesity is one of the major health problems throughout world. The present study investigated preventive effect epilactose – a rare non-digestible disaccharide on obesity and metabolic disorders in mice fed high-fat (HF) diets. Feeding with HF diets increased body weight gain, fat pad adipocyte size ( P <0·01), these increases were effectively prevented by use supplemental without influencing food intake <0·01). Caecal pools SCFA such as acetic propionic acids higher compared...

Enterohemorrhagic Escherichia coli (EHEC), causes a potentially life-threatening infection in humans worldwide. Serovar O157:H7, and to lesser extent serovars O26 O111, are the most commonly reported EHEC responsible for large number of outbreaks. We have established rapid discrimination method E. O157, O111 from other serovars, based on pattern matching mass spectrometry (MS) differences presence/absence biomarker proteins detected matrix-assisted laser desorption/ionization time-of-flight...

The genetic lineages of Listeria monocytogenes and other species the genus are correlated with pathogenesis in humans. Although matrix-assisted laser desorption ionization-time flight mass spectrometry (MALDI-TOF MS) has become a prevailing tool for rapid reliable microbial identification, precise discrimination remains crucial issue clinical settings food safety. In this study, we constructed an accurate MS database to discriminate L. (L. monocytogenes, innocua, welshimeri, seeligeri,...

We report a rapid and cost-effective monoclonal antibody screening method from single animal B cells using reverse transcription (RT)-PCR Escherichia coli cell-free protein synthesis (CFPS), which allows evaluation of antibodies within 2 working days. This process is named "Ecobody technology". The includes strategies to isolate that specifically bind an antigen the peripheral blood immunised animals, single-cell RT-PCR generate DNA fragments VH VL genes, followed by CFPS for production...

A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to C-terminus heavy chain (Hc, VH-CH1) and light (Lc, VL-CL), respectively, accelerate association Hc Lc form Escherichia coli vivo vitro expression systems. The...

Single B cell sampling following to direct gene amplification and transient expression in animal cells has been recognized as powerful monoclonal antibodies (mAbs) screening strategies. Here we report Ecobody technology which allows mAbs from single two days This uses Escherichia coli cell-free protein synthesis (CFPS) for mAb expression. In the CFPS step, employed our original techniques: (1) ‘Zipbody’ a modified Fab (fragment of antigen binding) format, active formation is facilitated by...

O157, O26, and O111 are the most important O serogroups of enterohemorrhagic Escherichia coli worldwide. Recently we reported a strategy for discriminating these serotypes from others using matrix-assisted laser desorption ionization-time flight mass spectrometry (MALDI-TOF MS) based on S10-spc-alpha operon gene-encoded ribosomal protein spectrum (S10-GERMS) method. To realize fully automated identification microorganisms at species- or serotype-level with concept S10-GERMS method, novel...

Abstract The rapid identification and classification of pathogenic microorganisms, including Salmonella enterica , is important for the surveillance prevention foodborne diseases. Matrix‐assisted laser desorption\ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) has been shown to be an effective tool microorganisms. In a previous report, database consisting 12 biomarker proteins, S8, L15, L17, L21, L25, S7, superoxide dismutase (SodA), peptidylprolyl cis‐trans isomerase C, Gns, YibT,...

The α-glucosidase HaG from the halophilic bacterium Halomonas sp. strain H11 catalyzes hydrolysis of glucosidic linkage at nonreducing end α-glucosides, such as maltose and sucrose, to release α-glucose. Based on its amino-acid sequence, this enzyme is classified a member glycoside hydrolase family 13. has three unique characteristics: (i) very narrow substrate specificity, almost exclusively hydrolyzing disaccharides; (ii) activation by monovalent cations, K(+), Rb(+), Cs(+) NH4(+); (iii)...

Abstract Ribosome arrest peptides (RAPs) such as SecM peptide (SecM AP) and WPPP with consecutive Pro residues, are known to induce translational stalling in Escherichia coli . We demonstrate that the translation enhancing SKIK tag, which consisted of four amino acid residues Ser-Lys-Ile-Lys, effectively alleviate caused by WPPP. Moreover, proximity between significantly influences extent this alleviation, observed both PURE cell-free protein synthesis vivo production systems, resulting a...

Abstract Immunoglobulins in the intestine have been reported to play a pivotal role regulating composition and function of intestinal microbiota. However, relationship between monoclonal antibodies (mAbs) bacteria remains elusive due lack analytical methods. Here we report utilization Ecobody technology, single B cell technique developed by our group, for obtaining investigating bacteria-binding mAbs derived from cells non-immunized rabbit. Bacteroides cellulosilyticus isolated rabbit stool...

A method has been developed for efficiently enriching and analyzing high-affinity antibody variants by combining the PURE ribosome display (ribosome using purified cell-free protein synthesis components) with next-generation sequencing (NGS) Brevibacillus choshinensis secretion system, NZ-1 antibody, which targets PA tag peptide (GVAMPGAEDDVV), as a model antibody. An artificial DNA encoding single-chain fragment of binding (scFab) was synthesized actively expressed system (CFPS)....

Ribosome arrest peptides (RAPs) such as the SecM peptide (SecM AP: FSTPVWISQAQGIRAGP) and WPPP with consecutive Pro residues are known to induce translational stalling in