Trapping SARS-CoV-2 main protease into transient artificial zymogens for high-yield expression and simplified purification.

During SARS-CoV-2 replication, the main protease (Mpro) autocatalytically cleaves itself from the viral polyprotein and forms an active dimer that processes the polyprotein into functional proteins. For full activity, Mpro must retain an authentic N-terminus. High-quality Mpro is critical for biochemical, structural, and antiviral studies. We compared three vector design strategies to express active Mpro. The first strategy employed autoprocessing fusion in which a SUMO domain is followed by the native Mpro cleavage site, Mpro, and a C-terminal HisTag, enabling efficient autocatalytic release of Mpro with an authentic N-terminus and a tagged C-terminus. The second strategy utilized external protease cleavage involving HisTag-SUMO-Mpro fusion, which requires ULP-1 protease to remove the N-terminal fusion, thereby restoring the authentic Mpro N-terminus along with enzymatic function. The third strategy leveraged a self-processing variant in which a HisTag-SUMO domain is linked to Mpro via a mutated cleavage site, allowing slow self-processing on Ni-NTA resin without exogenous protease or imidazole elution, thus reducing the number of purification steps. All three approaches yielded Mpro with similar N-termini, circular dichroism spectra, kinetic parameters, and thermal stability. The resulting proteins are suitable for biophysical studies and crystallization trials. These expression systems are potentially adaptable to other proteases whose activity is compromised by N-terminal extensions.