Monitoring of enzymatic cleavage reaction of GST-fusion protein on biolayer interferometry sensor.

Biophys Physicobiol

Faculty of Pharmaceutical Sciences, Hokkaido University of Science, Sapporo, Hokkaido 006-8585, Japan.

Published: September 2024

Biolayer interferometry (BLI) is an optical sensor-based analytical method primarily used for analyzing interactions between biomolecules. In this study, we explored the application of BLI to observe the cleavage reaction of glutathione S-transferase (GST)-tagged fusion protein by human rhinovirus (HRV) 3C protease on a BLI sensor as a new application of the BLI method. The soluble domain of the Tic22 protein from was expressed and purified as a GST-tagged fusion protein, GST-Tic22, in . A cleavage sequence for HRV 3C protease was inserted between the GST tag and the soluble domain of Tic22. First, we confirmed that GST-Tic22 was specifically cleaved at the inserted sequence by HRV 3C protease using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following this, GST-Tic22 was immobilized on a BLI sensor, and enzymatic cleavage by the HRV 3C protease was monitored. We observed that the soluble domain of Tic22 was cleaved and released into the buffer over time, and this reaction was dependent on the enzyme concentration. This result demonstrates that the BLI method can be used to evaluate the cleavage of the GST tag by the HRV 3C protease in real time under different conditions. This method enables a more efficient search for the optimal conditions for the tag cleavage reaction in fusion proteins, a process that has historically required a substantial amount of time and effort.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11718170PMC
http://dx.doi.org/10.2142/biophysico.bppb-v21.0019DOI Listing

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