研究者詳細 - 中野　秀雄

Development of a dual monoclonal antibody sandwich enzyme-linked immunosorbent assay for the detection of swine influenza virus using rabbit monoclonal antibody by Ecobody technology 査読有り

Sila-on, D; Chertchinnapa, P; Shinkai, Y; Kojima, T; Nakano, H

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 130 巻 ( 2 ) 頁： 217 - 225 2020年8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 dual monoclonal antibody sandwich enzyme-linked immunosorbent assay (mAb sandwich ELISA) has been developed using rabbit monoclonal antibodies generated by Ecobody technology, which includes the isolation of single B cells binding to a specific antigen, amplification of the heavy and light chains of these immunoglobulins, and expression of the fragment of antigen binding (Fab) by cell-free protein synthesis (CFPS). A rabbit was immunized with swine influenza virus (SIV) vaccine, from which single B cells binding to the antigen were isolated. Then, immunoglobulin mRNA was amplified from single cells by reverse transcription-polymerase chain reaction, followed by the attachment of a T7 promoter, appropriate tags, and a T7 terminator for the expression of the Fab portion by CFPS. By taking advantage of two different peptide tags fused to the same Fab, optimal combinations for coating Fab on assay plates and detecting Fab, both synthesized by CFPS, were investigated for mAb sandwich ELISA. Pairs of Fab detected 0.5 ng SIV in the assay. In summary, this result showed the applicability of Ecobody technology for a variety of immunodetection kits for high throughput analyses.

DOI： 10.1016/j.jbiosc.2020.03.003

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

Kojima, T; Nakane, A; Zhu, B; Alfi, A; Nakano, H

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 83 巻 ( 10 ) 頁： 1822 - 1828 2019年10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

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). The HRP activity was quantitatively detected on a BLI sensor chip by tracing a binding response of tyramide, a substrate of HRP, onto an immobilized protein. This system could be applied to analyses related to oxidase activity, as well as to the functional analysis of recombinant HRP.

DOI： 10.1080/09168451.2019.1621156

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

Alfi, A; Zhu, B; Damnjanovic, J; Kojima, T; Iwasaki, Y; Nakano, H

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 128 巻 ( 3 ) 頁： 290 - 295 2019年9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

Manganese peroxidase (MnP) is a fungal heme-containing enzyme which oxidizes Mn2+ to Mn3+, 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. Aiming to investigate the bacterial production of MnP, we have revealed an interesting mechanism underlying chaperone-assisted maturation of this enzyme to its active form. Since we previously found that in vitro expression of MnP in E. coli system depends on disulfide bond isomerase DsbC, we chose SHuffle T7 Express, an E. coli constitutively expressing DsbC, as the host for in vivo expression of MnP. Initially, only a low amount of the enzyme was present in the soluble fraction, with no detectable peroxidase activity. Co-expression of MnP with different chaperone revealed that DnaK, DnaJ, and GrpE contributed the most to the solubility improvement, however, remained in a complex with the MnP, preventing the enzyme to assume its active conformation. We resolved this by in vitro maturation, involving incubation of the MnP-chaperone complex with hemin, ATP, and ATP regeneration system. While ATP enables the chaperones to finish the refolding cycle and release the MnP in its correctly folded form, hemin supports the formation of the holo-enzyme with fully recovered peroxidase activity. We believe that the findings of this paper will serve as an important clue for establishing the bacterial production of fungal peroxidases in the future.

DOI： 10.1016/j.jbiosc.2019.02.011

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

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

Oka, H; Kojima, T; Ihara, K; Kobayashi, T; Nakano, H

BMC GENOMICS 20 巻 ( 1 ) 頁： 16 2019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：BMC Genomics

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. To date, the gene expression network controlled by AoXlnR in A. oryzae is not fully explored. In this study, the data from gSELEX-Seq analysis and data mining were applied toward a comprehensive investigation of the AoXlnR-regulated transcriptional network in A. oryzae. Results: Around 2000 promoters were selected as AoXlnR-binding DNAs using gSELEX-Seq, consequently identifying the genes downstream of them. On the other hand, 72 differentially expressed genes (DEGs) related to AoXlnR had been determined by microarray analysis. The intersecting set of genes, that were found using the gSELEX-Seq and the microarray analysis, had 51 genes. Further, the canonical AoXlnR-binding motifs, 5′-GGCT(A/G) A-3′, were successfully identified in gSELEX-Seq. The motif numbers in each promoter of the DEGs and differential expression levels were correlated by in silico analysis. The analysis showed that the presence of both 5′-GGCTAA-3′ and 5′-GGCTGA-3′ motif has significantly high correlation with the differential expression levels of the genes. Conclusions: Genes regulated directly by AoXlnR were identified by integrated mining of data obtained from gSELEX-Seq and microarray. The data mining of the promoters of differentially expressed genes revealed the close relation between the presence of the AoXlnR-binding motifs and the expression levels of the downstream genes. The knowledge obtained in this study can contribute greatly to the elucidation of AoXlnR-mediated cellulose and xylan metabolic network in A. oryzae. The pipeline, which is based on integrated mining of data consisting of both in vitro characterization of the DNA-binding sites and TF phenotype, can be a robust platform for comprehensive analysis of the gene expression network via the TFs.

DOI： 10.1186/s12864-018-5375-5

Damnjanovic, J; Nakano, H; Iwasaki, Y

PROTEIN ENGINEERING DESIGN & SELECTION 32 巻 ( 1 ) 頁： 1 - 11 2019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：Protein Engineering, Design and Selection

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. To meet this demand, we aimed at altering sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysoPLs, by protein engineering. Based on the crystal structure of Streptomyces antibioticus PLD, W166 was targeted for saturation mutagenesis due to its strong interaction with the sn-2 acyl chain of the PL. Screening result pointed at W166R and W166K PLDs to selectively react on lysophosphatidylcholine (lysoPC), while not on PC. These variants showed a negative correlation between activity and sn-2 chain length of PL substrates. This behavior was not observed in the wild-type (WT)-PLD. Kinetic analysis revealed that the W166R and W166K variants have 7-10 times higher preference to lysoPC compared to the WT-PLD. Additionally, W166R PLD showed detectable activity toward glycero-3-phosphocholine, unlike the WT-PLD. Applicability of the lysoPC-preferring PLD was demonstrated by detection of lysoPC in the mixed PC/lysoPC sample and by the synthesis of cyclic phosphatidic acid. Structure model analyses supported the experimental findings and provided a basis for the structure model-based hypothesis on the observed behavior of the enzymes.

DOI： 10.1093/protein/gzz019

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

Ritthisan, P; Ojima-Kato, T; Damnjanovic, J; Kojima, T; Nakano, H

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 126 巻 ( 6 ) 頁： 705 - 709 2018年12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

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. Our results demonstrated that the SKIK-Fab-LZ-AP fusion was well expressed in E. coli oxidative cytoplasm in soluble form having both antigen-binding and AP activity, and was purified to homogeneity by two step column chromatography, suggesting that the combination of the SKIK tag and AP fusion can greatly increase the productivity and solubility of the Fab-enzyme fusion in an E. coli cytoplasmic expression system.

DOI： 10.1016/j.jbiosc.2018.06.009

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

Kojima, T; Hata, J; Oka, H; Hayashi, K; Hitomi, K; Nakano, H

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 82 巻 ( 11 ) 頁： 1911 - 1921 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：Bioscience, Biotechnology and Biochemistry

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. Furthermore, these DNA-binding tags were used to establish an enzyme assay system based on the spatial arrangement of transglutaminase and its substrate at the molecular level. Together, the results of the present study suggest that the scCro-tag may be a powerful tool to facilitate the synthetic spatial arrangement of proteins on a DNA ligand.

DOI： 10.1080/09168451.2018.1501265