TY - JOUR
T1 - Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays
AU - Kojima, Takaaki
AU - Mizoguchi, Takuro
AU - Ota, Eri
AU - Hata, Jumpei
AU - Homma, Keisuke
AU - Zhu, Bo
AU - Hitomi, Kiyotaka
AU - Nakano, Hideo
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
UR - http://www.scopus.com/inward/record.url?scp=84953361942&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84953361942&partnerID=8YFLogxK
U2 - 10.1016/j.jbiosc.2015.06.003
DO - 10.1016/j.jbiosc.2015.06.003
M3 - Article
C2 - 26522987
AN - SCOPUS:84953361942
VL - 121
SP - 147
EP - 153
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
SN - 1389-1723
IS - 2
ER -