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In Vitro GT-array (i-GT-ray), a Platform for Screening of Glycosyltransferase Activities and Protein-Protein
Matrika Bhattarai1,2, Tasleem Javaid1, Akshayaa Venkataraghavan1
1Department of Environmental and Plant Biology, Ohio University, Athens, OH, USA.
Bio-Protocol
|September 30, 2024
Summary
Researchers developed the in vitro GT-array (i-GT-ray) platform for rapid screening of glycosyltransferase (GT) enzyme activity and protein-protein interactions (PPIs). This innovative tool accelerates biochemical characterization, overcoming limitations of traditional methods.
Area of Science:
- Biochemistry
- Molecular Biology
- Bioinformatics
Background:
- Numerous glycosyltransferases (GTs) identified via bioinformatics have unknown functions due to technical limitations.
- Current methods for assessing GT enzyme activity and protein-protein interactions (PPIs) are time-consuming and not high-throughput.
Purpose of the Study:
- To develop an innovative platform for rapid, large-scale screening of GT enzyme activity and PPIs.
- To provide a user guide for the in vitro GT-array (i-GT-ray) platform.
Main Methods:
- Developed the i-GT-ray platform using 96-well microplates coated with plasmid DNA and capture antibodies.
- Utilized cell-free in vitro transcription/translation (IVTT) to synthesize tagged GTs directly from plasmid DNA.
- Captured synthesized GTs via anti-tag antibodies on microplates, enabling direct enzyme activity and PPI assays.
Main Results:
- The i-GT-ray platform allows for the synthesis, capture, and purification of tagged GTs in under two days.
- Enables high-throughput screening of enzyme activity and PPIs for GTs.
- Facilitates biochemical characterization of challenging proteins, including membrane-integrated GTs.
Conclusions:
- The i-GT-ray platform significantly accelerates the functional characterization of glycosyltransferases.
- This method overcomes previous technical hurdles, enabling efficient large-scale screening and PPI analysis.
- The platform is adaptable for various analytical techniques, including mass spectrometry.
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