Part:BBa_K5131009

pGEX-GST-nsp5_T21I-His

Information of SARS-Cov-2 nsp5 can be seen in BBa_K5131000. After characterization of BBa_K5131007, we found kcat/Km of nsp5 (27,691 s⁻¹M⁻¹) is much higher than TEV protease((kcat/Km =2,600 s⁻¹M⁻¹)) and HRV 3C protease(kcat/Km =840 s⁻¹M⁻¹) which is commonly used for removal of recombinant tags during protein purification.Thus we thought nsp5 may be better suited for this purpose so we aimed to enhance its enzymatic activity through rational design to make nsp5 can be used as a tool enzyme. We rational design a mutant nsp5-T21I which incresed the kcat/Km from 27,691 s⁻¹M⁻¹ to 35,069 s⁻¹M⁻¹ . Our work confirms that this mutant is more suitable as a tool enzyme compared to the wild type. We hope that this part can be a low-cost and efficient tag removal tool compared to directly purchasing commercial proteases for recombinant tag removal.

Rational design of SARS-Cov-2 nsp5

Our strategy was to introduce mutations in nsp5 that could strengthen its binding affinity to the linker substrate (N-GSAVLQSGFRK-C), thereby increaseing nsp5's activity. Given that the catalytic center of the enzyme is relatively conserved, mutations in the catalytic core often lead to loss of function. Therefore, we chose to modify amino acids that are relatively distant from the catalytic center but still involved in substrate binding. Additionally, to facilitate comparisons of interactions before and after mutation, we focused on amino acids with relatively simple interactions with the substrate. First, we predicted the structure of the wild-type nsp5 in complex with the linker substrate. Through structural analysis, we found that T21_nsp5 is distant from the catalytic center and interacts with only one amino acid of the substrate. Therefore, we chose to modify this site. To enhance the interaction between T21_nsp5and the substrate, we aimed to replace T21_nsp5 with an amino acid that has a more extended side chain, while retaining the original characteristics of the side chain. For this purpose, we chose to mutate T to I. This mutation replaces the hydroxyl group attached to the carbon atom of the R-group with a -CH₂-CH₃group, increasing the side chain's length. We then predicted the structure of the nsp5-T21I mutant in complex with the same linker substrate and performed a comparative analysis with the wild-type nsp5. The results showed that the overall structures of the two were very similar (Cα RMSD = 0.16), with only the R10 residue of the substrate(R10_substrate) exhibiting a rotation of approximately 50 degrees. Therefore, we focused on analyzing this region in detail (Figure 1).

Figure 1. Structural comparison of the wild-type nsp5 and nsp5 T21I in complex with the linker substrate. Wild-type nsp5 is shown in green, while nsp5-T21I is shown in cyan.

By comparing the structures, we found that when T21_nsp5 was replaced by I21_nsp5, the R10_substrate side chain, which previously interacted with T21_nsp5, was displaced from its original position. Although the interaction with residue 21 was weakened, this subtle conformational change allowed the carbonyl oxygen of G23_nsp5 to form additional interactions with the R10_substrate side chain. Additionally, the distance between the carbonyl oxygens of T24_nsp5 and R10_substrate decreased from 2.8 Å to 2.7 Å.

Figure 2. Interaction between wild-type nsp5 and nsp5-T21I with the R10_substrate, with an interaction distance threshold of 4 Å and the interaction indicated by yellow dashed lines. Wild-type nsp5 is shown in green, while nsp5-T21I is shown in cyan.

In summary, in wild-type nsp5, only T24_nsp5 and T21_nsp5 interact with R10_substrate, whereas in nsp5-T21I, residues I21_nsp5, G23_nsp5, and T24_nsp5 together stabilize R10_substrate. We hypothesize that this change enhances the interaction between the substrate and nsp5, leading to improved enzymatic activity.

Construction of pGEX-GST-nsp5_T21I-His

We first successfully amplified the vector backbone and the nsp5_native-6His tag separately using PCR (Figure 2B). Subsequently, we constructed the pGEX-GST-nsp5_native-His through homologous recombination. The sequencing results confirmed the correct construction of our vector(Figure 2B).

Figure 3. A) Vector design of pGEX-GST-nsp5_T21I-His. B) Sequencing validation of nsp5_T21I.

Express validation and characterization nsp5_T21I

We expressed the protein in E. coli BL21 and purified it using Ni-NTA affinity chromatography. Protein expression was induced by adding IPTG to a final concentration of 0.2 mM.SDS-PAGE indicated that nsp5-T21I had high purity and a molecular weight consistent with expectations(Figure 4).This suggests that the nsp5-T21I has native N- and C-terminus.

Figure 4.Purification of nsp5-T21I. Lane 1-2: marker, purified protein.

To validate our design, we subsequently measured the enzymatic activity of nsp5_T21I using FRET as mentioned in BBa_K5131007. 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 and recorded the changes in fluorescence intensity with an plate reader. After the reaction started, we measured the fluorescence intensity every two seconds. By linearly fitting the fluorescence intensity for the first 40 s, we obtained reaction rates of 0.0497, 0.1351, 0.3698, 0.5414, and 0.7573 for nsp5-T21I at substrate concentrations of 2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM, respectively. Compared with nsp5, nsp5-T21I reacted faster at the same substrate concentration, suggesting that nsp5-T21I cleaves linker substrates more efficiently than nsp5(Figure 5).

Figure 5. Reaction rates comparsion between WT nsp5 and nsp5 T21I.

We further applied the Michaelis-Menten equation to fit the reaction rates at different substrate concentrations, resulting in the determination of Km and Kcat values for nsp5-T21I. The result shows that the kcat/Km of nsp5-T21I is 35,069 s⁻¹M⁻¹(Figure 6).The enzyme activity of nsp5-T21I was increased by 26.6% compared to wild-type nsp5(kcat/Km=27,691 s⁻¹M⁻¹), suggesting that nsp5-T21I has more potential to be developed as a new tool enzyme.

Figure 6. Kinetic model of nsp5-T21I.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1738
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 770
  Illegal XhoI site found at 1733
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1177
  Illegal SapI.rc site found at 167