Expression, purification, and biophysical characterization of recombinant MERS-CoV main (Mpro) protease

Ghada Obeid Almutairi; Ajamaluddin Malik; Mona Alonazi; Javed Masood Khan; Abdullah S Alhomida; Mohd Shahnawaz Khan; Amal M Alenad; Nojood Altwaijry; Nouf Omar Alafaleq

doi:10.1016/j.ijbiomac.2022.04.077

Expression, purification, and biophysical characterization of recombinant MERS-CoV main (M^pro) protease

Int J Biol Macromol. 2022 Jun 1;209(Pt A):984-990. doi: 10.1016/j.ijbiomac.2022.04.077. Epub 2022 Apr 19.

Authors

Ghada Obeid Almutairi¹, Ajamaluddin Malik², Mona Alonazi¹, Javed Masood Khan³, Abdullah S Alhomida¹, Mohd Shahnawaz Khan⁴, Amal M Alenad¹, Nojood Altwaijry⁴, Nouf Omar Alafaleq¹

Affiliations

¹ Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia.
² Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia. Electronic address: amalik@ksu.edu.sa.
³ Department of Food Science and Nutrition, Faculty of Food and Agricultural Sciences, King Saud University, 2460, Riyadh 11451, Saudi Arabia.
⁴ Protein Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.

Abstract

MERS-CoV main protease (M^pro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV M^pro under different conditions is poorly characterized. In this study, MERS-CoV M^pro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV M^pro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV M^pro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of M^pro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV M^pro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 M^pro shows 96.08% and 50.65% similarity to that of SARS-CoV M^pro and MERS-CoV M^pro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV M^pro.

Keywords: Differential scanning fluorometry; MERS-CoV; Molten globule.

MeSH terms

Coronavirus 3C Proteases* / genetics
Coronavirus 3C Proteases* / metabolism
Middle East Respiratory Syndrome Coronavirus* / enzymology
Middle East Respiratory Syndrome Coronavirus* / genetics
Protein Conformation
Recombinant Proteins

Substances

Recombinant Proteins
Coronavirus 3C Proteases