Effect of Translation-Enhancing Nascent SKIK Peptide on the Arrest Peptides Containing Consecutive Proline.

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

Construction of transcript regulation mechanism prediction models based on binding motif environment of transcription factor AoXlnR in .

DNA-binding transcription factors (TFs) play a central role in transcriptional regulation mechanisms, mainly through their specific binding to target sites on the genome and regulation of the expression of downstream genes. Therefore, a comprehensive analysis of the function of these TFs will lead to the understanding of various biological mechanisms. However, the functions of TFs are diverse and complicated, and the identified binding sites on the genome are not necessarily involved in the regulation of downstream gene expression.

Substrate profiling of human transglutaminase 1 using cDNA display and next-generation sequencing.

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences.

Rapid and cost-effective epitope mapping using PURE ribosome display coupled with next-generation sequencing and bioinformatics.

A novel, efficient and cost-effective approach for epitope identification of an antibody has been developed using a ribosome display platform. This platform, known as PURE ribosome display, utilizes an Escherichia coli-based reconstituted cell-free protein synthesis system (PURE system). It stabilizes the mRNA-ribosome-peptide complex via a ribosome-arrest peptide sequence.

Nascent MSKIK peptide cancels ribosomal stalling by arrest peptides in Escherichia coli.

The insertion of the DNA sequence encoding SKIK peptide adjacent to the M start codon of a difficult-to-express protein enhances protein production in Escherichia coli. In this report, we reveal that the increased production of the SKIK-tagged protein is not due to codon usage of the SKIK sequence. Furthermore, we found that insertion of SKIK or MSKIK just before the SecM arrest peptide (FSTPVWISQAQGIRAGP), which causes ribosomal stalling on mRNA, greatly increased the production of the protein containing the SecM arrest peptide in the E.

Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics.

cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (10) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3.

Administration of suppresses DSS-induced colitis.

, a filamentous fungus, has long been used for the production of traditional Japanese foods. Here, we analyzed how administration affects the intestinal environment in mice. The results of 16S rRNA gene sequencing of the gut microbiota indicated that after the administration of heat-killed spores, the relative abundance of an anti-inflammatory strain became 2.

Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: insights into plasticity of substrate binding and activation.

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesise a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised.

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins.

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs.

Rapid Generation of Monoclonal Antibodies from Single B Cells by Ecobody Technology.

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 screening from single B cells in two days This technology uses cell-free protein synthesis (CFPS) for mAb expression. In the CFPS step, we employed our original techniques: (1) 'Zipbody' as a modified Fab (fragment of antigen binding) format, in which the active Fab formation is facilitated by adhesive leucine zipper peptides fused at the C-termini of the light and heavy chains; and (2) an N-terminal SKIK peptide tag that can markedly increase protein production.

Acyl chain that matters: introducing sn-2 acyl chain preference to a phospholipase D by protein engineering.

Phospholipase D (PLD) is an enzyme widely used for enzymatic synthesis of structured phospholipids (PLs) with modified head groups. These PLs are mainly used as food supplements and liposome ingredients. Still, there is a need for an enzyme that discriminates between PLs and lysoPLs, for specific detection of lysoPLs in various specimens and enzymatic synthesis of certain PLs from a mixed substrate.

A simple, real-time assay of horseradish peroxidase using biolayer interferometry.

Horseradish peroxidase (HRP) isoenzyme C1a is one of the most widely used enzymes for various analytical methods in bioscience research and medical fields. In these fields, real-time monitoring of HRP activity is highly desirable because the utility of HRP as a reporter enzyme would be expanded. In this study, we developed a simple assay system enabling real-time monitoring of HRP activity by using biolayer interferometry (BLI).

Production of active manganese peroxidase in Escherichia coli by co-expression of chaperones and in vitro maturation by ATP-dependent chaperone release.

Manganese peroxidase (MnP) is a fungal heme-containing enzyme which oxidizes Mn to Mn, a diffusible and strong non-specific oxidant capable of attacking bulky phenolic substrates. Therefore, MnP is indispensable in the polymer and paper industries. Previous attempts of MnP expression in Escherichia coli resulted in the formation of inclusion bodies which required in vitro refolding.

Comprehensive investigation of the gene expression system regulated by an Aspergillus oryzae transcription factor XlnR using integrated mining of gSELEX-Seq and microarray data.

Background: Transcription factors (TFs) specifically bind to DNA sequences and control the expression of target genes. AoXlnR is a key TF involved in the expression of xylanolytic and cellulolytic enzymes in the filamentous fungi, Aspergillus oryzae. Genomic SELEX-Seq (gSELEX-Seq) can reveal the in vitro binding sites of a TF in a genome.

Spatial arrangement of proteins using scCro-tag: application for an in situ enzymatic microbead assay.

In natural systems, various metabolic reactions are often spatially organized to increase enzyme activity and specificity. Thus, by spatially arranging enzyme molecules in synthetic systems to imitate these natural systems, it is possible to promote a high rate of enzymatic turnover. In this present study, a normal and mutant form of the scCro DNA-binding protein were shown to bind orthogonally to specific recognition sequences under appropriate conditions.

SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasm.

Antibody-enzyme fusion proteins have been used for various immunological detection techniques, such as ELISA, Western blotting and so on. The use of genetically-fused antibody-enzyme complexes has advantages over conventional chemical conjugation methods, as they require no complex chemical reactions and allow for the strict control of the number of enzymes fused with antibodies, resulting in a more stable performance of the bifunctional protein. Here, we describe efficient cytoplasmic soluble expression of an antigen-binding fragment (Fab) fused with Escherichia coli alkaline phosphatase (AP), N-terminal Ser-Lys-Ile-Lys (SKIK) tag that can improve the synthesis of the tagged protein, as well as leucine zipper (LZ) to enhance the association of the light chain and the heavy chain of Fab.

Zipbodyzyme: Development of new antibody-enzyme fusion proteins.

A new antibody-enzyme fusion protein, named Zipbodyzyme, composed of a Fab antibody (i.e., an antigen-binding fragment of an antibody) and an enzyme, has been successfully produced in the cytoplasm of Escherichia coli.

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation.

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

Development of a rapid immunoassay system: Luminescent detection of antigen-associated antibody-luciferase in the presence of a dye that absorbs light from free antibody-luciferase.

In this report, we developed a rapid immunoassay system, designated the bioluminescent interference gathering optical (BINGO) assay, which required no time-consuming washing steps for removal of unbound antibodies. This system employed a luciferase (Luc)-conjugated antibody (LucAb) and a dye that absorbed light from the LucAb. The antigen-associated LucAb was localized by transfer of an antigen to the detector-side of a chamber where a detector photomultiplier tube (PMT) was installed.

Strain improvement of Lentzea sp. 7887 for higher yield per unit volume on hydroxylation of cyclosporine derivative FR901459.

A Gram-positive bacterium Lentzea sp. 7887 hydroxylates a cyclosporine derivative FR901459 into AS1837812 (9-hydroxide), which is an important intermediate of candidate drugs that target the hepatitis C virus. We screened a UV-induced mutant, named M-1, which showed about 1.

N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae.

Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E.

Pilot-scale whole-cell biocatalysis for the hydroxylation of cyclosporine derivative, FR901459, at higher concentrations by Lentzea sp. 7887 using soybean flour as a novel substrate dispersant.

Pilot-scale hydroxylation of FR901459, an immunosuppressive cyclosporine derivative, was performed using resting cells of a Gram-positive bacteria Lentzea sp. 7887 (as whole-cell biocatalysts) and soybean flour as a substrate dispersant. Through biocatalysis, FR901459 was hydroxylated at position 9, producing AS1837812, an important intermediate in the production of drug candidates against hepatitis C.