Faculty Profiles - NAKANO, Hideo

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 Reviewed

Daorung Sila-on, Phornnaphat Chertchinnapa, Yusuke Shinkai, Takaaki Kojima, Hideo Nakano

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 130 ( 2 ) page： 217 - 225 2020.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

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. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2020.03.003

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

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

Takaaki Kojima, Ayako Nakane, Bo Zhu, Almasul Alfi, Hideo Nakano

Bioscience, Biotechnology, and Biochemistry Vol. 83 ( 10 ) page： 1822 - 1828 2019.10

Almasul Alfi, Bo Zhu, Jasmina Damnjanović, Takaaki Kojima, Yugo Iwasaki, Hideo Nakano

Journal of Bioscience and Bioengineering Vol. 128 ( 3 ) page： 290 - 295 2019.9

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

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

BMC genomics Vol. 20 ( 1 ) page： 16 2019.1

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

Jasmina Damnjanović, Hideo Nakano, Yugo Iwasaki

Protein Eng. Des. Sel. Vol. 32 ( 1 ) page： 1 - 11 2019.1

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

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

Panwad Ritthisan, Teruyo Ojima-Kato, Jasmina Damnjanovic, Takaaki Kojima, Hideo Nakano

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 126 ( 6 ) page： 705 - 709 2018.12

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

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. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2018.06.009

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

Kojima Takaaki, Hata Jumpei, Oka Hiroya, Hayashi Kenta, Hitomi Kiyotaka, Nakano Hideo

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Vol. 82 ( 11 ) page： 1911 - 1921 2018.11

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

ABSTRACT

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