Design

Expression, purification and characterization of SARS-CoV-2 nsp5

1. Construction of nsp5 expression vectors

To achieve the goal of the screening platform, the first step of this large process is to express a functional nsp5 in E. coli. To make sure the successful expression of the SARS-CoV-2 nsp5 , we selected the pGEX-6p-1 vector that includes a GST tag and a HRV 3C protease cleavage site. Considering the purification of the protein, we fused a 6*His tag to the C-terminal of the nsp5 gene sequence. We also introduced four amino acids (AVLQ) at the N-terminus of nsp5 to facilitate its self-cleavage from the GST tag^[1], resulting in nsp5 with both native N- and C-termini (see details in Engineering Success). We named it pGEX-GST-nsp5_native-His(Figure 1).

2. Expression and purification of SARS-CoV-2 nsp5

We expressed the protein in E. coli BL21 and purified it using Ni-NTA affinity chromatography. First, BL21 competent cells transformed with the pGEX-GST-nsp5_native-His plasmid were cultured at 37°C until the OD reached ~0.6. Then, protein expression was induced by adding IPTG to a final concentration of 0.2 mM. After 16 hours of induction, the cells were lysed, and the supernatant was collected by high-speed centrifugation (13,000 xg). The supernatant was passed through a Ni-NTA affinity column to allow the target protein to bind to the Ni-NTA beads. After repeatedly rinsing the beads with 20 mM imidazole-containing buffer until the eluate did not make the G250 turn blue, HRV 3C protease was added to remove the excess amino acids from the N- and C-termini of the target protein, separating it from the Ni-NTA beads. Finally, the purified SARS-CoV-2 nsp5 was eluted and concentrated for further analysis(Figure 2).

3. Characterization of SARS-CoV-2 nsp5

In this section, we aimed to assess the activity of nsp5 to confirm that the nsp5 we expressed in E.coli is functional. Additionally, as a new iGEM part, we aim to characterize it in as much detail as possible.

For this purpose, we used the fluorescence resonance energy transfer (FRET) technique to assess the enzyme activity^[2]. We designed a fluorescent probe, MCA-AVLQS GFRK (DnP) K, which can be recognized and cleaved by nsp5 as a substrate. When nsp5 fails to cleave the probe, the donor (MCA) and acceptor (DnP) group remain in proximity. This causes FRET to occur, which leads to a reduction of fluorescence. On the contrary, when the probe is cleaved, the donor and acceptor groups move away from each other, disrupting FRET and emitting fluorescence. Thus, we can characterize the activity of nsp5 cleavage substrates through the changes in fluorescence intensity.

The reaction process of substrate cleavage by nsp5 (Figure 4) can be described by the classical Michaelis-Menten equation. We aimed to determine the reaction rate of nsp5 at different substrate concentrations to characterize its Km and kcat in detail. We fixed the concentration of nsp5 at 1.2 µM and the substrate concentrations at 2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM, and carried out the reaction at 30 °C, recording the changes in fluorescence intensity with an plate reader. After the reaction started, we measured the fluorescence intensity every two seconds. The fluorescence intensity for the first 40 s was fitted by a linear equation, the slope of which is the rate of change of fluorescence intensity, indicating the reaction rate of nsp5. After obtaining the reaction speed of nsp5 at different substrate concentrations, we used the Michaelis-Menten equation to finally obtain the K_m and k_cat of nsp5.

In vivo inhibitor screening platform based on FlipGFP

In order to detect the activity of nsp5 protease and to verify the inhibitory effect of exogenous inhibitors on it, we plan to monitor protein interactions using a novel fluorescent protein, FlipGFP^[3], which consists of 11 β-strands and a central α-helix. 10th and 11th β-strands by redesigning them so that they form a parallel structure before the action of the protease and thus cannot self-assemble with β-strands 1-9. In FlipGFP, the 11th β-strand is linked to a protease cleavage sequence. After cleavage, the 11th β-strand reverts to an antiparallel structure, allowing GFP to self-assemble and emit fluoresce. We plan to construct a FlipGFP with a specially engineered recognition sequence using the specific cleavage sequence of the nsp5 (AVLQSFGRK) linked to the 11th β-strand. After using this split fluorescent protein to bind to the nsp5 protease vector and characterizing its enzyme activity by fluorescence intensity, our screening system will be very efficient due to FlipGFP's high brightness, and fast response. We also hope to introduce a uniform cleavage site at both ends of the recognition sequence of each viral protease, so that different recognition sequences can be quickly replaced by a simple enzymatic reaction, thus making the platform more flexible and facilitating the study of different viral proteases.

To ensure the correct expression of FlipGFP, we used the pRSF-Duet1 and separately inserted the first nine β-strands of FlipGFP and the engineered 10-11 β-strands into two different ORFs. To facilitate inhibitor screening, we designed a genetic circuit with the following functionalities: when only FlipGFP is present, the entire system does not emit fluorescence; when both nsp5 and FlipGFP are present, nsp5 cleaves FlipGFP, resulting in fluorescence; and when nsp5 is inhibited, the cleavage efficiency of nsp5 on FlipGFP decreases, leading to reduced or absent fluorescence.

Rational design to enhance nsp5 enzymatic activity

TEV protease(K_m/k_cat =2,600 s⁻¹M⁻¹)^[4] and HRV 3C protease (K_m/k_cat =840 s⁻¹M⁻¹)^[5] are commonly used tool enzymes for the removal of recombinant tags. Since nsp5 has higher efficiency(K_m/k_cat =27,691 s⁻¹M⁻¹), we thought nsp5 may be better suited for this purpose. Thus, we aimed to enhance its enzymatic activity through rational design to allow nsp5 to be used as a tool enzyme.Our strategy was to introduce mutations in nsp5 that could strengthen its binding affinity to the linker substrate (N-GSAVLQSGFRK-C), thereby increasing nsp5's activity. We used alphafold3 to predict the structure of the nsp5 mutant-substrate complex and compared it with the wild type (see Model for details). For mutants that may have enhanced affinity, we subsequently performed expression purification and measured changes in enzyme activity.

References

1. Jin Z, Du X, Xu Y, Deng Y, Liu M, Zhao Y, Zhang B, Li X, Zhang L, Peng C, Duan Y, Yu J, Wang L, Yang K, Liu F, Jiang R, Yang X, You T, Liu X, Yang X, Bai F, Liu H, Liu X, Guddat LW, Xu W, Xiao G, Qin C, Shi Z, Jiang H, Rao Z, Yang H. Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors. Nature. 2020 Jun;582(7811):289-293. doi: 10.1038/s41586-020-2223-y. Epub 2020 Apr 9. PMID: 32272481.
2. Tripathi PK, Upadhyay S, Singh M, Raghavendhar S, Bhardwaj M, Sharma P, Patel AK. Screening and evaluation of approved drugs as inhibitors of main protease of SARS-CoV-2. Int J Biol Macromol. 2020 Dec 1;164:2622-2631. doi: 10.1016/j.ijbiomac.2020.08.166. Epub 2020 Aug 24. PMID: 32853604; PMCID: PMC7444494.
3. Zhang Q, Schepis A, Huang H, Yang J, Ma W, Torra J, Zhang SQ, Yang L, Wu H, Nonell S, Dong Z, Kornberg TB, Coughlin SR, Shu X. Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals. J Am Chem Soc. 2019 Mar 20;141(11):4526-4530. doi: 10.1021/jacs.8b13042. Epub 2019 Mar 6. PMID: 30821975; PMCID: PMC6486793.
4. Parks TD, Howard ED, Wolpert TJ, Arp DJ, Dougherty WG. Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form. Virology. 1995 Jun 20;210(1):194-201. doi: 10.1006/viro.1995.1331. PMID: 7793070.
5. Wang QM, Johnson RB. Activation of human rhinovirus-14 3C protease. Virology. 2001 Feb 1;280(1):80-6. doi: 10.1006/viro.2000.0760. PMID: 11162821.