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

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

doi:10.1016/j.jbiosc.2015.06.003

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

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

Biotechnology Institute

Research output: Contribution to journal › Article › peer-review

10 Scopus citations

Abstract

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding O_R consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.

Original language	English (US)
Pages (from-to)	147-153
Number of pages	7
Journal	Journal of Bioscience and Bioengineering
Volume	121
Issue number	2
DOIs	https://doi.org/10.1016/j.jbiosc.2015.06.003
State	Published - Feb 1 2016

Bibliographical note

Funding Information:
This work was supported in part by a Grant-in-Aid Scientific Research (B) (no. 23360367 ) and Grant-in-Aid for Scientific Research on Innovative Areas aimed at Synthetic Biology for the Comprehension of Biological Networks (no. 26119707 ) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) .

Publisher Copyright:
© 2015 The Society for Biotechnology, Japan.

Keywords

Cell-free protein synthesis
Flow cytometry
Manganese peroxidase
OR consensus DNA
ScCro
Transglutaminase 2

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10.1016/j.jbiosc.2015.06.003

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title = "Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays",

abstract = "A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.",

keywords = "Cell-free protein synthesis, Flow cytometry, Manganese peroxidase, OR consensus DNA, ScCro, Transglutaminase 2",

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

note = "Funding Information: This work was supported in part by a Grant-in-Aid Scientific Research (B) (no. 23360367 ) and Grant-in-Aid for Scientific Research on Innovative Areas aimed at Synthetic Biology for the Comprehension of Biological Networks (no. 26119707 ) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) . Publisher Copyright: {\textcopyright} 2015 The Society for Biotechnology, Japan.",

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doi = "10.1016/j.jbiosc.2015.06.003",

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AU - Kojima, Takaaki

AU - Mizoguchi, Takuro

AU - Ota, Eri

AU - Hata, Jumpei

AU - Homma, Keisuke

AU - Zhu, Bo

AU - Hitomi, Kiyotaka

AU - Nakano, Hideo

N1 - Funding Information: This work was supported in part by a Grant-in-Aid Scientific Research (B) (no. 23360367 ) and Grant-in-Aid for Scientific Research on Innovative Areas aimed at Synthetic Biology for the Comprehension of Biological Networks (no. 26119707 ) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) . Publisher Copyright: © 2015 The Society for Biotechnology, Japan.

PY - 2016/2/1

Y1 - 2016/2/1

N2 - A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.

AB - A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.

KW - Cell-free protein synthesis

KW - Flow cytometry

KW - Manganese peroxidase

KW - OR consensus DNA

KW - ScCro

KW - Transglutaminase 2

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

Abstract

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