研究者詳細 - 中野　秀雄

An improved laser photo-detachment diagnostic for negative ion density measurement

Rattanawongnara E., Nakano H., Tsumori K., Nagaoka K., Osakabe M.

Journal of Instrumentation 19 巻 ( 11 ) 2024年11月

出版者・発行元：Journal of Instrumentation

A photo-detachment Langmuir probe is a crucial tool because it gives a point measurement of negative ion density. The detection circuitry of a photo-detachment diagnostic with nanosecond laser pulses is critical for the accuracy of the results. Applying the electromagnetic theory to the design of the photo-detachment system has allowed it to stabilize its frequency response up to -445 MHz, providing a significantly higher time resolution than in a common photo-detachment circuit setup. A systematic design rule is given in this paper to standardize the proper circuit. The new standard allows comparison between laboratories without concern for electronic parameter differences. The high-time resolution result shows three different peaks in the photo-detached electron current. This paper identified that the first peak is the most correlated to negative-ion density information, and the second and third peaks are related to background electrons interacting with build-up potential.

DOI： 10.1088/1748-0221/19/11/P11002

The Identification of a Target Gene of the Transcription Factor KojR and Elucidation of Its Role in Carbon Metabolism for Kojic Acid Biosynthesis in Aspergillus oryzae Open Access

Mizutani, T; Oka, H; Goto, R; Tsurigami, R; Maruyama, J; Shimizu, M; Kato, M; Nakano, H; Kojima, T

JOURNAL OF FUNGI 10 巻 ( 2 ) 2024年2月

記述言語：英語出版者・発行元：Journal of Fungi

DNA-binding transcription factors are broadly characterized as proteins that bind to specific sequences within genomic DNA and modulate the expression of downstream genes. This study focused on KojR, a transcription factor involved in the metabolism of kojic acid, which is an organic acid synthesized in Aspergillus oryzae and is known for its tyrosinase-inhibitory properties. However, the regulatory mechanism underlying KojR-mediated kojic acid synthesis remains unclear. Hence, we aimed to obtain a comprehensive identification of KojR-associated genes using genomic systematic evolution of ligands by exponential enrichment with high-throughput DNA sequencing (gSELEX-Seq) and RNA-Seq. During the genome-wide exploration of KojR-binding sites via gSELEX-Seq and identification of KojR-dependent differentially expressed genes (DEGs) using RNA-Seq, we confirmed that KojR preferentially binds to 5′-CGGCTAATGCGG-3′, and KojR directly regulates kojT, as was previously reported. We also observed that kojA expression, which may be controlled by KojR, was significantly reduced in a ΔkojR strain. Notably, no binding of KojR to the kojA promoter region was detected. Furthermore, certain KojR-dependent DEGs identified in the present study were associated with enzymes implicated in the carbon metabolic pathway of A. oryzae. This strongly indicates that KojR plays a central role in carbon metabolism in A. oryzae.

DOI： 10.3390/jof10020113

Bio-nanocapsules for oriented immobilization of DNA aptamers on aptasensors 査読有り

Iijima, M; Yamada, Y; Nakano, H; Nakayama, T; Kuroda, S

ANALYST 147 巻 ( 3 ) 頁： 489 - 495 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Analyst

The oriented immobilization of sensing molecules (e.g., IgGs, receptors, lectins, and DNA aptamers) on sensor chips is particularly important for maximizing the potential of the sensing molecules, thereby enhancing the sensitivity and target-binding capacity of biosensors. We previously developed ∼30 nm bio-nanocapsules (ZZ-BNCs) consisting of the hepatitis B virus envelope L protein fused with the tandem form of protein A-derived IgG Fc-binding Z domain (ZZ-L protein). ZZ-BNC acts successfully as a scaffold, enhancing both the sensitivity and binding capacity of IgG, a Fc-fused receptor, and Fc-fused lectin to antigens, cytokines, and sugar chains through an oriented immobilization on a biosensor surface. To expand the versatility of ZZ-BNC, we modified ZZ-BNC by replacing the ZZ domain with a DNA-binding single-chain lambda Cro (scCro) domain, thereby developing scCro-BNC. The scCro-BNC was synthesized in yeast cells and homogeneously purified as ∼30 nm sized nanoparticles. In a quartz crystal microbalance, an scCro-BNC-coated sensor chip immobilized with thrombin-binding DNA aptamers showed an ∼5.5-fold higher thrombin-binding capacity and ∼6000-fold higher detection sensitivity than a sensor chip directly coated with DNA aptamers. In addition, the number of bound thrombin molecules per molecule of DNA aptamer increased by ∼7.8-fold with an scCro-BNC coating, consistent with the theoretical thrombin-binding capacity. Collectively, scCro-BNC was shown to perform as an ideal scaffold for maximizing the potential of the DNA aptamer by immobilizing it in an oriented manner. Facilitating a highly sensitive detection of various target molecules, these BNC-based scaffolds are expected to improve a wide range of biosensors while minimizing the number of sensing molecules required. This journal is

DOI： 10.1039/d1an02278d

Development of robust and rapid monoclonal antibody screening technology using cell-free protein synthesis

無細胞タンパク質合成系を利用した迅速抗体スクリーニング技術開発と実用化招待有り

加藤晃代, 中野秀雄, 兒島孝明, 永井里美

日本生物工学会会誌 99 巻 ( 2 ) 頁： 62 - 67 2021年2月

Ojima-Kato T., Nakano H., Kojima T., Nagai S.

Seibutsu-kogaku Kaishi 99 巻 ( 2 ) 頁： 62 - 67 2021年

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

Ojima-Kato T, Morishita S, Uchida Y, Nagai S, Kojima T, Nakano H

Antibodies (Basel, Switzerland) 7 巻 ( 4 ) 2018年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Antibodies

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 Escherichia coli 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. By the Ecobody technology, we demonstrated rapid screening of antigen specific mAbs from immunized rabbits and Epstein-Barr Virus infected human B cells. We further obtained rabbit mAbs in E. coli expression system yielding to 8.5 mg of purified proteins from 1 L bacterial culture.

DOI： 10.3390/antib7040038

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

Nishikawa, Y; Fujikawa, R; Nakano, H; Kanamori, T; Ojima-Kato, T

ACS SYNTHETIC BIOLOGY 13 巻 ( 12 ) 頁： 3908 - 3916 2024年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACS Synthetic Biology

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 Escherichia coli. 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. Furthermore, we unveil that nascent SKIK peptide tag and translation elongation factor P (EF-P) alleviate ribosome stalling in consecutive-Pro-rich protein to synergistically promote translation. A kinetic analysis based on the generation of superfolder green fluorescent protein under in vitro translation reaction reveals that the ribosome turnover is enhanced by more than 10-fold when the SKIK peptide tag is positioned immediately upstream of the SecM AP sequence. Our findings provide valuable insights into optimizing protein production processes, which are essential for advancing synthetic biology applications.

DOI： 10.1021/acssynbio.4c00221

Construction of transcript regulation mechanism prediction models based on binding motif environment of transcription factor AoXlnR in Aspergillus oryzae 国際誌

Oka, H; Kojima, T; Kato, R; Ihara, K; Nakano, H

JOURNAL OF BIOINFORMATICS AND COMPUTATIONAL BIOLOGY 22 巻 ( 03 ) 頁： 2450017 - 2450017 2024年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bioinformatics and Computational Biology

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 in vivo are diverse and complicated, and the identified binding sites on the genome are not necessarily involved in the regulation of downstream gene expression. In this study, we investigated whether DNA structural information around the binding site of TFs can be used to predict the involvement of the binding site in the regulation of the expression of genes located downstream of the binding site. Specifically, we calculated the structural parameters based on the DNA shape around the DNA binding motif located upstream of the gene whose expression is directly regulated by one TF AoXlnR from Aspergillus oryzae, and showed that the presence or absence of expression regulation can be predicted from the sequence information with high accuracy (AUPRC=0.8-1.0) by machine learning incorporating these parameters.

DOI： 10.1142/S0219720024500173

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

Substrate profiling of human transglutaminase 1 using cDNA display and next-generation sequencing 査読有り Open Access

Munaweera, TIK; Damnjanovic, J; Camagna, M; Nezu, M; Jia, BX; Hitomi, K; Nemoto, N; Nakano, H

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 88 巻 ( 6 ) 頁： 620 - 629 2024年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioscience, Biotechnology and Biochemistry

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. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.

DOI： 10.1093/bbb/zbae029

その他リンク： https://academic.oup.com/bbb/article-pdf/88/6/620/57805618/zbae029.pdf

Jia, BX; Ojima-Kato, T; Kojima, T; Nakano, H

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 137 巻 ( 4 ) 頁： 321 - 328 2024年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bioscience and Bioengineering

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. This system was complemented by next-generation sequencing (NGS) and an algorithm for analyzing binding epitopes. To showcase the effectiveness of this method, selection conditions were refined using the anti-PA tag monoclonal antibody with the PA tag peptide as a model. Subsequently, a random peptide library was constructed using 10 NNK triplet oligonucleotides via the PURE ribosome display. The resulting random peptide library-ribosome-mRNA complex was selected using a commercially available anti-HA (YPYDVPDYA) tag monoclonal antibody, followed by NGS and bioinformatic analysis. Our approach successfully identified the DVPDY sequence as an epitope within the hemagglutinin amino acid sequence, which was then experimentally validated. This platform provided a valuable tool for investigating continuous epitopes in antibodies.

DOI： 10.1016/j.jbiosc.2024.01.008

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

Ojima-Kato, T; Nishikawa, Y; Furukawa, Y; Kojima, T; Nakano, H

JOURNAL OF BIOLOGICAL CHEMISTRY 299 巻 ( 5 ) 頁： 104676 - 104676 2023年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

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. coli–reconstituted cell-free protein synthesis system (PURE system). A similar translation enhancement phenomenon by MSKIK was observed for the CmlA leader peptide, a ribosome arrest peptide, whose arrest is induced by chloramphenicol. These results strongly suggest that the nascent MSKIK peptide prevents or releases ribosomal stalling immediately following its generation during the translation process, resulting in an increase of protein production.

DOI： 10.1016/j.jbc.2023.104676