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

Paper ID Volume ID Publish Year Pages File Format Full-Text
20012 43153 2016 7 PDF Available
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
scCro; OR consensus DNA; Transglutaminase 2; Manganese peroxidase; Flow cytometry; Cell-free protein synthesis
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Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 121, Issue 2, February 2016, Pages 147–153
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
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Price after discount Only $4.95
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