Contribution

Overview

In this year’s project, we focused on developing an in vivo inhibitor screening platform targeting SARS-CoV-2 nsp5, contributing five new basic parts and four new composite parts to the iGEM community. Our main contributions can be summarized in the following key areas:

  • We successfully expressed and purified the main drug target of SARS-CoV-2, nsp5, and characterized its enzymatic activity parameters(BBa_K5131001).
  • We successfully designed FlipGFP that can be activated by nsp5, contributing effective protease-responsive parts to the iGEM community(BBa_K5131008).
  • We performed further rational design on nsp5 to enhance its enzymatic activity, increasing its potential for broader applications (BBa_K5131002).

Expression, purification, and enzymatic activity characterization of nsp5 with native N- and C-termini

Nsp5 is a crucial target for SARS-CoV-2, and developing an efficient platform for screening nsp5 inhibitors can provide a foundation for controlling COVID-19 as well as offer insights for screening protease inhibitors of other positive-strand RNA viruses. Therefore, at the initial stage of this project, our goal was to obtain highly pure, correctly folded, and highly active nsp5.

After two rounds of DBTL (as detailed in Engineering Success), we ultimately selected pGEX-6P-1 as the expression vector. We obtained the wild-type SARS-CoV-2 nsp5 sequence (NC_045512) from NCBI, inserted an auto-cleavage sequence AVLQ↓SGFR at its N-terminus, and fused a 6×His tag at its C-terminus for subsequent protein purification.

Figure 1. A) Vector design of pGEX-GST-nsp5_native-His. B) PCR amplification of nsp5_native and pGEX-6P-1. C) Sequencing validation of nsp5_native.
We expressed nsp5 in E. coli BL21 and purified it using nickel affinity chromatography (see Design for details). Due to the presence of the N-terminal auto-cleavage sequence, the nsp5 obtained has native N- and C-termini, with no residual amino acids that could potentially affect its activity.We subsequently characterized the enzymatic activity of nsp5 using FRET and confirmed that the nsp5 obtained from E. coli exhibits high enzymatic efficiency(Km/kcat =27,691 s⁻¹M⁻¹).
Figure 2. Purification of nsp5_native. Lane 1-3: marker, digested nickel beads, purified protein(left panel). Kinetic model of nsp5 enzyme activity(right panel).
In this part of the work, we contributed a well-characterized part, SARS-CoV-2 nsp5(BBa_K5131001), to the iGEM community. This part can be used by any iGEM team dedicated to researching SARS-CoV-2 therapies. Additionally, our cloning design that preserves the native N- and C-termini can serve as a reference for other iGEM teams involved in the purification of viral proteases.

Design of an in vivo nsp5 inhibitor screening platform based on FlipGFP

We aim to use FlipGFP to respond to the cleavage activity of nsp5, that is, to utilize nsp5 to activate FlipGFP to emit fluorescence and thus build an in vivo nsp5 inhibitor screening platform (see Design for details). Previously, Wageningen_UR attempted to construct a FlipGFP recognized by MMP-9 in 2022. They successfully created and submitted the part (BBa_K4244045), but unfortunately, their FlipGFP was not activated by MMP-9 and did not emit fluorescence.

We carefully examined the sequence of the part they submitted and found that FlipGFP is located within only one ORF, and there are subtle differences between the sequence information and those reported in the literature. To obtain a FlipGFP that can be correctly activated, we used the sequence reported in the original literature, replacing the TEV protease recognition sequence with the nsp5 recognition sequence. Additionally, we inserted the β-strands 1-9 and the engineered β-strands 10-11 into the two ORFs of pRSF-Duet1, respectively.

Figure 3. A) Vector design of pRSF-FlipGFP_nsp5(10-11)-FlipGFP(1-9). B) Sequencing validation of FlipGFP_nsp5(10-11)-FlipGFP(1-9).

By co-transforming nsp5 with FlipGFP, we found that our designed FlipGFP can be successfully activated by nsp5 to emit fluorescence, while FlipGFP alone does not produce fluorescence.

Figure 4. Activation of FlipGFP by nsp5

In summary, in this part of the project, we successfully designed a FlipGFP(BBa_K5131008) that can be activated by nsp5 and aim to apply it for inhibitor screening. Since detecting other protease activities only requires replacing a small segment of the recognition sequence, our work provides an effective tool for in vivo protease activity detection for other iGEM teams. Previous applications of FlipGFP were primarily focused on eukaryotic systems; our work also demonstrates the feasibility of FlipGFP in prokaryotic systems. Finally, our work offers new insights for in vivo protease inhibitor screening.

Rational design to enhance nsp5 enzymatic activity

In the protein purification process, removing recombinant tags is often necessary to ensure the protein remains in its native state. Currently, some proteases used for tag removal are also derived from viruses, such as HRV 3C protease and TEV protease. In preliminary experiments, we found that the enzymatic activity of nsp5 is significantly higher than that of TEV protease and HRV 3C protease. Therefore, our goal is to enhance the enzymatic activity of nsp5 through rational design to improve its effectiveness for recombinant tag removal.

Based on structural predictions and comparative analysis, we proposed a mutation that could potentially enhance enzymatic activity: nsp5-T21I (see Model for details). Subsequently, we successfully expressed and purified nsp5-T21I in E. coli BL21 and verify its higher efficiency(Km/kcat=35,069 s⁻¹M⁻¹) than WT nsp5.

Figure 5. Kinetic model of nsp5-T21I(left panel). Reaction rates comparsion between WT nsp5 and nsp5 T21I (right panel).

This part of the work primarily aims to provide the iGEM community with a new tool enzyme for protein purification. The removal of recombinant tags is a crucial step in protein purification, and we offer a more efficient new tool(BBa_K5131002) for this process. Additionally, the purification process of nsp5 is relatively straightforward. After nickel affinity chromatography, high-purity protein suitable for subsequent experiments can be obtained without the need for further fine purification. This approach significantly reduces the cost of this step compared to purchasing commercial proteases.