A dual-tag strategy with superfolder green fluorescent protein (sfGFP) and small ubiquitin-like modifier (SUMO) for soluble expression of SARS-CoV-2 RNA polymerase visibly in Escherichia coli.

Publication date: Jul 13, 2025

RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 composed of three non-structural proteins NSP12, NSP8, and NSP7, which is responsible for replication and transcription, making it a promising target for antiviral drug development. However, the limited solubility presents a significant challenge in the expression of NSP12, restricting in-depth research in both scientific and clinical fields. To address the challenge of soluble expression of NSP12 using the bacterial expression system, we developed a strategy integrating the super-folder GFP (sfGFP) tag to overcome the low solubility of NSP12 in this study. By engineering an sfGFP-mediated auto-secretion platform that eliminates signal peptides while enabling real-time visualization, we achieved soluble production of all RdRp subunits (NSP12/8/7) using a two-step nickel-affinity purification strategy in E. coli. Furthermore, the tandem sfGFP-SUMO fusion strategy and optimized expression conditions (18 ^0C, 0. 5 mM IPTG) were used to enhance the efficient expression of NSP12, further boosting the yield of NSP12 to 1. 73 mg/L. This produced all soluble RdRp subunits (NSP12/8/7), with size-exclusion chromatography confirming complex assembly, offering a universal framework for other important insoluble viral protein production. The integration of real-time visualization with hyper-solubility of this system addresses both the technical barrier in viral protein expression and the urgent need for scalable antiviral drug development platforms.

Semantics