Recombinant full-length 2019-nCoV 3CLpro, part of a large replicase polyprotein 1ab (S3264-Q3569), was expressed in E. coli cells with an N-terminal His tag.  The purity of the protein was determined to be approx. 90% by densitometry with a molecular weight of 35 kDa.

Our 3CLpro protease activity is detected using FRET-based assay using Edans/Dabcyl peptide substrate, in an intact FRET peptide, the fluorescence of Edans is quenched due to its close proximity to the Dabcyl quencher.

Upon the cleavage in two separate fragments by the protease, the fluoresces is recovered, and can be monitored at an excitation wavelength of 340nm/495nm​

SARS-CoV-2 3C-like proteinase (3CLpro) is the nonstructural protein number 5, also a cysteine protease, that is released from the virally encoded polyprotein by extensive proteolytic processing.

The 3CLpro cleaves the C-terminus of the large replicase polyprotein 1ab (~790 kDa) as much as 11 sites.1 Inhibiting the activity of this enzyme would block viral replication .2

The functional importance of 3CLpro in the viral life cycle makes this protease an attractive target for the development of drugs directed against SARS and other coronavirus infections.3

Product specifications

Specific activity

Sample Activity Plot.

The protease specific activity of 3CLpro was determined to be 4170 pmol/min/mg at an enzyme concentration of 1.2 μm as per the activity assay protocol. Sample Activity Plot. Image Credit: SignalChem Biotech

For particular data on a given lot, users can refer the related technical data sheet.

Purity

Sample Purity Data.

Sample Purity Data. Image Credit: SignalChem Biotech

For particular data on a given lot, users can refer the related technical data sheet.

Storage, stability, and shipping

The product should be stored at –70 °C. For optimal storage, the target should be aliquoted into smaller quantities after centrifugation and then stored at suggested temperature. For most favorable performance, multiple freeze/thaw cycles and  frequent handling should be avoided.

Molecular weight

35 kDa

References

  1. Hegyi, A. et al: Conservation of substrate specificities among coronavirus main proteases. J Gen Virol. 2002,83:595-599.
  2. Zhang L. et al: Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science. 2020, 368:409-412.
  3. Anand K. et al: Coronavirus main proteinase (3CLpro) structure: Basis for design of anti-SARS drugs. Science. 2003, 300:1763-1767.