Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays
| Autor: | Hideo Nakano, Takaaki Kojima, Keisuke Homma, Kiyotaka Hitomi, Bo Zhu, Takuro Mizoguchi, Eri Ota, Jumpei Hata |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Repressor Bioengineering Protein tag Biology Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound GTP-Binding Proteins Consensus Sequence Protein biosynthesis A-DNA Protein Glutamine gamma Glutamyltransferase 2 Viral Regulatory and Accessory Proteins Enzyme Assays chemistry.chemical_classification Cell-free protein synthesis Transglutaminases 030102 biochemistry & molecular biology Cell-Free System Substrate (chemistry) DNA Enzymes Immobilized Flow Cytometry Microspheres DNA-Binding Proteins Repressor Proteins 030104 developmental biology Enzyme Biochemistry chemistry Peroxidases Biocatalysis Biotechnology |
| Zdroj: | Journal of bioscience and bioengineering. 121(2) |
| ISSN: | 1347-4421 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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