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

Research output: Contribution to journalArticlepeer-review

7 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 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.

Original languageEnglish (US)
Pages (from-to)147-153
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume121
Issue number2
DOIs
StatePublished - Feb 1 2016

Keywords

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

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