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.
Bibliographical noteFunding 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) .
© 2015 The Society for Biotechnology, Japan.
Copyright 2016 Elsevier B.V., All rights reserved.
- Cell-free protein synthesis
- Flow cytometry
- Manganese peroxidase
- OR consensus DNA
- Transglutaminase 2