Part:BBa_K5131007

pGEX-GST-nsp5_native-His

Information of SARS-Cov-2 nsp5 can be seen in BBa_K5131000. After learning from BBa_K5131006, we added 4 four amino acids (AVLQ) at the N-terminus of nsp5 to produce nsp5 with native N- and C- terminus. We use this part to express and purify SARS-Cov-2 nsp5 in native state and verified its activity (Km/kcat=27,691 s⁻¹M⁻¹).

Sequence design of pGEX-GST-nsp5_nativte-His

Based on previous experience, we introduced four amino acids (AVLQ) at the N-terminus of nsp5 to facilitate its self-cleavage from the GST tag. The rest of the sequence remained consistent with BBa_K5131006 (Figure 1).

Figure 1. Sequence design for GST-nsp5_native-His. ”↓“for the cleavage site.

Construction of pGEX-GST-nsp5_nativte-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 2C).

Figure 2. A) Vector design of pGEX-GST-nsp5-His. B) PCR amplification of nsp5_native and pGEX-6P-1. C) Sequencing validation of nsp5.

Express validation nsp5_native

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_native had high purity and a molecular weight consistent with expectations(Figure 3).This suggests that the nsp5_native is the SARS-Cov-2 nsp5 with native N- and C-terminus.

Figure 3. Purification of nsp5. Lane 1-8: marker, control group, supernatant after centrifugation, nickel beads before digested, flow-through buffer, eluted buffer, digested nickel beads, purified protein

Characterization nsp5_native

For characterization of nsp5_native, we used the fluorescence resonance energy transfer (FRET) technique to assess the enzyme activity. 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, and thus FRET occurs leading to quenching of fluorescence. On the contrary, when the probe is cleaved, the donor and acceptor groups move away from each other, thus disrupting FRET and emitting fluorescence. Thus we can characterize the activity of nsp5 cleavage substrates by changes in fluorescence intensity. Since 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.

Figure 4. The mechanism of substrate cleavage by nsp5.

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. 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 and can also be regarded as the reaction rate of nsp5(Figure 5).

Figure 5. Linear fitting of fluorescent intensity over the first 40 seconds of the reaction.

By linear fitting, we obtain that the reaction rate of nsp5 is 0, 0.1089, 0.2560, 0.5109 and 0.6222 when the substrate concentration is 2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM, respectivelyFinally, we applied the Michaelis-Menten equation to fit the reaction velocities at different substrate concentrations, resulting in the determination of Km and Kcat values for nsp5 (Figure 6).

Figure 6. Kinetic model of nsp5 enzyme activity.

In summary, we derived the kinetic equation for nsp5:

The catalytic efficiency of nsp5 was determined to be Kcat/Km =27,691 s⁻¹M⁻¹ .

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