Faculty Profiles - NAKANO, Hideo

Papers - NAKANO, Hideo

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Leucine Zipper fused Fab; Enhancement of active Fab formation in E. coli in vitro and in vivo expression systems Reviewed

Ojima-Kato Teruyo, Fukui Kansuke, Kojima Takaaki, Nakano Hideo

PROTEIN SCIENCE Vol. 24 page： 194-194 2015.10

Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter Reviewed

Zhu Bo, Mizoguchi Takuro, Kojima Takaaki, Nakano Hideo

PLOS ONE Vol. 10 ( 5 ) 2015.5

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays Reviewed

Kojima Takaaki, Mizoguchi Takuro, Ota Eri, Hata Jumpei, Homma Keisuke, Zhu Bo, Hitomi Kiyotaka, Nakano Hideo

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 121 ( 2 ) page： 147-153 2016.2

In vitro generation of rabbit anti-Listeria monocytogenes monoclonal antibody using single cell based RT-PCR linked cell-free expression systems Reviewed

Ojima-Kato Teruyo, Hashimura Dai, Kojima Takaaki, Minabe Shiori, Nakano Hideo

JOURNAL OF IMMUNOLOGICAL METHODS Vol. 427 page： 58-65 2015.12

Construction of a DNA Library on Microbeads Using Whole Genome Amplification Reviewed

Kojima Takaaki, Zhu Bo, Nakano Hideo

WHOLE GENOME AMPLIFICATION: METHODS AND PROTOCOLS Vol. 1347 page： 87-100 2015

Evaluation of antibody variants using a ribosome display and Brevibacillus choshinensis secretion system.

Kihara M, Okuda R, Okada A, Ojima-Kato T, Nakano H

Journal of bioscience and bioengineering 2025.3

Language：English Publisher：Journal of Bioscience and Bioengineering

In antibody engineering, the development of rapid and efficient strategies for improving affinity is highly necessary. In this study, we aimed to establish a method to efficiently enrich and analyze high-affinity antibody variants by combining protein synthesis using recombinant elements (PURE) ribosome display with next-generation sequencing (NGS) and Brevibacillus choshinensis secretion system using the NZ-1 antibody, which targets the PA tag peptide (GVAMPGAEDDVV) as a model antibody. From the mutated scFab library designed based on the structure, we performed a single-round of PURE ribosome display selection and analyzed the data by NGS to obtain high-affinity scFab candidates with high enrichment factor and high read counts. Subsequently, the most promising candidate was produced as a Fab in the B. choshinensis secretion system, and the purified Fab had an affinity (KD = 1.6 × 10−9 M) similar to the wild type. Overall, this study highlights the potential of the integrated PURE ribosome display with NGS analysis and the B. choshinensis secretion system for the rapid identification and analysis of high-affinity antibody variants.

DOI： 10.1016/j.jbiosc.2025.02.009

Scopus

PubMed

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

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

Journal of Instrumentation Vol. 19 ( 11 ) 2024.11

Publisher：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

Scopus

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

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

JOURNAL OF FUNGI Vol. 10 ( 2 ) 2024.2

Language：English Publisher：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 Reviewed

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

ANALYST Vol. 147 ( 3 ) page： 489 - 495 2022.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Web of Science

Scopus

PubMed

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 Invited

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 Vol. 99 ( 2 ) page： 62 - 67 2021.2

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

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

Seibutsu-kogaku Kaishi Vol. 99 ( 2 ) page： 62 - 67 2021

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

Ojima-Kato Teruyo, Morishita Shiomi, Uchida Yoshino, Nagai Satomi, Kojima Takaaki, Nakano Hideo

ANTIBODIES Vol. 7 ( 4 ) 2018.11

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

Yuma Nishikawa, Riko Fujikawa, Hideo Nakano, Takashi Kanamori, Teruyo Ojima-Kato

ACS Synthetic Biology Vol. 13 ( 12 ) page： 3908 - 3916 2024.11

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

Hiroya Oka, Takaaki Kojima, Ryuji Kato, Kunio Ihara, Hideo Nakano

Journal of bioinformatics and computational biology Vol. 22 ( 03 ) page： 2450017 - 2450017 2024.6

Language：English Publishing type：Research paper (scientific journal)

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 ([Formula: see text]-1.0) by machine learning incorporating these parameters.

DOI： 10.1142/S0219720024500173

Web of Science

Scopus

PubMed

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

T I K Munaweera, Jasmina Damnjanović, Maurizio Camagna, Moeri Nezu, Beixi Jia, Kiyotaka Hitomi, Naoto Nemoto, Hideo Nakano

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY Vol. 88 ( 6 ) page： 620 - 629 2024.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

Abstract

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

Other Link： https://academic.oup.com/bbb/article-pdf/88/6/620/57805618/zbae029.pdf

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

Beixi Jia, Teruyo Ojima-Kato, Takaaki Kojima, Hideo Nakano

Journal of bioscience and bioengineering Vol. 137 ( 4 ) page： 321 - 328 2024.4

Language：English Publishing type：Research paper (scientific journal)

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

Web of Science

Scopus

PubMed

Nascent MSKIK Peptide Cancels Ribosomal Stalling by Arrest Peptides in Escherichia coli

Teruyo Ojima-Kato, Yuma Nishikawa, Yuki Furukawa, Takaaki Kojima, Hideo Nakano

Journal of Biological Chemistry Vol. 299 ( 5 ) page： 104676 - 104676 2023.5

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

Jasmina Damnjanović, Nana Odake, Jicheng Fan, Maurizio Camagna, Beixi Jia, Takaaki Kojima, Naoto Nemoto, Kiyotaka Hitomi, Hideo Nakano

Scientific Reports Vol. 12 ( 1 ) page： 13578 2022.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Abstract

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<sup>12</sup>) 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. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.

DOI： 10.1038/s41598-022-17494-4

Other Link： https://www.nature.com/articles/s41598-022-17494-4

Administration of Aspergillus oryzae suppresses DSS-induced colitis. International journal Open Access

Ryo Nomura, Sho Tsuzuki, Takaaki Kojima, Mao Nagasawa, Yusuke Sato, Masayoshi Uefune, Yasunori Baba, Toshiya Hayashi, Hideo Nakano, Masashi Kato, Motoyuki Shimizu

Food chemistry. Molecular sciences Vol. 4 page： 100063 - 100063 2022.7

Language：English Publishing type：Research paper (scientific journal)

Aspergillus oryzae, a filamentous fungus, has long been used for the production of traditional Japanese foods. Here, we analyzed how A. oryzae 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 A. oryzae spores, the relative abundance of an anti-inflammatory Bifidobacterium pseudolongum strain became 2.0-fold greater than that of the control. Next, we examined the effect of A. oryzae spore administration on the development of colitis induced by dextran sodium sulfate in mice; we found that colitis was alleviated by not only heat-killed A. oryzae spores, but also the cell wall extracted from the spores. Our findings suggest that A. oryzae holds considerable potential for commercial application in the production of both traditional Japanese fermented foods and new foods with prebiotic functions.

DOI： 10.1016/j.fochms.2021.100063

Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: Insights into plasticity of substrate binding and activation. Invited Reviewed International coauthorship

Samantha, A., Damnjanović, J., Iwasaki, Y., Nakano, H., and Vrielink, A.

Biochemical Journal Vol. 478 page： 1749 - 1767 2021.5

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Papers - NAKANO, Hideo