Optimization of the Chemical Monoubiquitination System for Low-Solubility Protein: Achieving Balance Between...

Monoubiquitination is a significant post-translational modification that plays a pivotal role in various biological processes. Chemical monoubiquitination holds significant value in investigating the functional implications of site-specific ubiquitination on target proteins. Despite all progress made in this area, conventional enzymatic methods so far rely largely on high yields of substrate proteins and the removal of tags to prevent non-specific ubiquitin binding, which poses substantial challenges for low-solubility proteins. Here, an optimized chemical monoubiquitination system that facilitates precise, site-specific ubiquitination of low-solubility target protein was developed using SSPP as an example. A cysteine-free GST tag (GST^4CS) was engineered, and a flexible (GGGGS)₃ linker was incorporated to mitigate steric hindrance and enhance the solubility of GST-SSPP fusion protein, resulting in a 2.5-fold increase in purification yield. Successful monoubiquitination of SSPP at the position of lysine 305 was achieved using disulfide-mediated conjugation, as proven via SDS-PAGE and Western blotting. Moreover, the phosphatase assay showed that monoubiquitination at residue C305 of the mutated SSPP significantly decreased its phosphatase activity. This system eliminates tag interference and enhances compatibility with low-solubility targets, providing a robust platform for functional studies of plant protein ubiquitination.