SARS-CoV-2 3CLpro as an efficient endopeptidase for fusion tag removal in recombinant protein production
Year: 2026
Type: article
Abstract: Fusion tags are widely employed to enhance recombinant protein expression, solubility, and purification. However, efficient tag removal is often necessary to obtain proteins in their native form. In this study, SARS-CoV-2 3C-like protease (3CLpro) was evaluated as a sequence-specific protease for fusion-tag removal using a recombinant fusion protein substrate under standard protein purification conditions. A substrate containing the canonical recognition sequence AVLQ↓SG was efficiently cleaved by 3CLpro, whereas no detectable cleavage was observed for a variant substrate harboring the sequence TVRLQ↓SG. Biochemical characterization demonstrated that 3CLpro maintained cleavage activity across a range of pH values and temperatures and in the presence of reagents commonly used for protein purification and stabilization. 3CLpro-mediated cleavage was successfully integrated into different fusion-protein processing workflows, including in-solution cleavage, on-column cleavage, and dialysis-coupled cleavage. To further evaluate the practical utility of 3CLpro, a modular expression vector (pET_RPLa) incorporating a 3CLpro cleavage site was constructed and validated using the light chain of botulinum neurotoxin serotype A (LC-BoNT/A) as a model protein. Following expression and purification, the fusion protein (6 × His-GST-AVLQSG-LC-BoNT/A) retained SNAP25-cleaving activity and was efficiently processed by 3CLpro. Together, these results support the potential use of SARS-CoV-2 3CLpro as a versatile sequence-specific protease for fusion-tag removal and recombinant protein processing.
Source: PLoS ONE
Institution: VNU University of Science
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,382
OpenAlex ID: W7225787277