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

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

Ghada Obeid Almutairi  1 , Ajamaluddin Malik  2 , Mona Alonazi  1 , Javed Masood Khan  3 , Abdullah S Alhomida  1 , Mohd Shahnawaz Khan  4 , Amal M Alenad  1 , Nojood Altwaijry  4 , Nouf Omar Alafaleq  1
Affiliations

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

Ghada Obeid Almutairi et al. Int J Biol Macromol. .

Abstract

MERS-CoV main protease (Mpro) 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 Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro 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 Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro 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 Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro.

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

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Figures

Fig. 1
Fig. 1
Purification of His-tagged MERS-CoV Mpro. Lane 1, marker; lane 2, total cell lysate; lane 3, flow-through; lane 4, wash; lane 5, fraction 1.
Fig. 2
Fig. 2
Intrinsic fluorescence spectra of MERS-CoV Mpro. (A) Emission spectra of MERS-CoV Mpro at different pHs. An excitation wavelength of 280 nm was used and monitored emission in the range of 300–400 nm. (B) λmax plotted with respect to pH.
Fig. 3
Fig. 3
Extrinsic fluorescence spectra of MERS-CoV Mpro. (A) Binding of ANS with MERS-CoV Mpro at different pHs. Samples were excited at 385 nm, and the spectra were recorded from 400 to 650 nm. (B) Imax plotted with respect to pH.
Fig. 4
Fig. 4
Analysis of aggregate formation in MERS-CoV Mpro at pH 1.0–7.0.by RLS. MERS-CoV Mpro excited at 350 nm and emission spectra recorded between 300 and 400 nm.
Fig. 5
Fig. 5
The thermal shift assay for MERS-CoV Mpro at pH 1.0–7.0. MERS-CoV Mpro samples were continuously heated from 20 to 90 °C at 1 °C min−1 and the spectra were collected at the range of 310–360 nm. The ratio of 350 nm/330 nm was plotted as a function of temperature.
Fig. 6
Fig. 6
Far UV CD studies of MERS-CoV Mpro. Far-UV CD spectra (200-250 nm) of 75 μg ml−1 MERS-CoV Mpro at pH 1.0 to 7.0.
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