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A fluorescence-based glycosyltransferase assay for high-throughput screening.
Jihye Ryu1, Min Sik Eom2, Wooseok Ko1
1Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea.
Bioorganic & Medicinal Chemistry
|March 22, 2014
Summary
A novel assay system for glycosyltransferases was developed using an ATP sensor. This sensitive and versatile system aids in high-throughput screening for glycosyltransferase engineering and drug discovery.
Area of Science:
- Biochemistry and Molecular Biology
- Enzymology
- Drug Discovery
Background:
- Glycosyltransferases are crucial enzymes catalyzing stereospecific sugar transfer.
- Engineering glycosyltransferases is key for diversifying carbohydrate-based drugs.
- High-throughput screening requires efficient glycosyltransferase assay systems.
Purpose of the Study:
- To develop a general assay system for glycosyltransferases.
- To enable high-throughput screening of glycosyltransferase mutants.
- To facilitate glycosyltransferase engineering for drug diversification.
Main Methods:
- Development of a novel assay system utilizing an ATP sensor.
- Validation of the assay system with purified enzymes.
- Testing the assay system's compatibility with crude cell extracts.
Main Results:
- The developed assay system demonstrated submicromolar sensitivity.
- The system proved compatible with both purified enzymes and crude cell extracts.
- The assay provides a robust platform for screening glycosyltransferase activity.
Conclusions:
- A versatile and sensitive glycosyltransferase assay system has been established.
- This assay system is suitable for high-throughput screening in glycosyltransferase engineering.
- The system supports general glycosyltransferase assays and kinetic studies.
