Difference between revisions of "Part:BBa K5131001"
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Information of SARS-CoV-2 nsp5 can be seen in <bbpart>BBa_K5131000</bbpart>. We utilises pGEX-6P-1 to express this parts to get nsp5 in its native state and verified its activity (Km/kcat=27,691 s⁻¹M⁻¹) through FRET. This part together with <bbpart>BBa_K5131008</bbpart> can be used as an in vivo inhibitor screening platform for nsp5.  
 
Information of SARS-CoV-2 nsp5 can be seen in <bbpart>BBa_K5131000</bbpart>. We utilises pGEX-6P-1 to express this parts to get nsp5 in its native state and verified its activity (Km/kcat=27,691 s⁻¹M⁻¹) through FRET. This part together with <bbpart>BBa_K5131008</bbpart> can be used as an in vivo inhibitor screening platform for nsp5.  
 
<h2> <b> Sequence design of pGEX-GST-nsp5_nativte-His </b> </h2>
 
<h2> <b> Sequence design of pGEX-GST-nsp5_nativte-His </b> </h2>
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In SARS-CoV-2 lifecycle, nsp5 must undergo self-cleavage at the N-terminus to release itself from the polyprotein[1]. After learning from <bbpart>BBa_K5131000</bbpart>, we introduced four amino acids (AVLQ) at the N-terminus of nsp5 to facilitate its self-cleavage from the GST tag, resulting in naive N- and C-termini. The rest of the sequence remained consistent with <bbpart>BBa_K5131000</bbpart> . (Figure 1).
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In SARS-CoV-2 lifecycle, nsp5 must undergo self-cleavage at the N-terminus to release itself from the polyprotein<sup>[1]</sup>. After learning from <bbpart>BBa_K5131000</bbpart>, we introduced four amino acids (AVLQ) at the N-terminus of nsp5 to facilitate its self-cleavage from the GST tag, resulting in naive N- and C-termini. The rest of the sequence remained consistent with <bbpart>BBa_K5131000</bbpart> . (Figure 1).
 
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<b>Figure 2. </b>A) Vector design of pGEX-GST-nsp5-His. B) PCR amplification of nsp5_native and pGEX-6P-1. C) Sequencing validation of nsp5.
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<b>Figure 2. </b>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.
 
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<h2> <b> Express validation nsp5_native </b> </h2>
 
<h2> <b> Express validation nsp5_native </b> </h2>
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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.  
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We expressed the protein in <i>E. coli</i> 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-termini.  
 
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<b>Figure 3. </b>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
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<b>Figure 3. </b>Purification of nsp5. Lane 1-3: marker, digested nickel beads, purified protein
 
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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, respectively. Finally, 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).
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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, respectively. Finally, we applied the Michaelis-Menten equation to fit the reaction velocities at different substrate concentrations, resulting in the determination of <I>K<sub>m</sub></i> and <i><sub>kcat</sub></i> values for nsp5 (Figure 6).
 
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Revision as of 13:31, 2 October 2024


SARS-Cov-2 nsp5_native

Information of SARS-CoV-2 nsp5 can be seen in BBa_K5131000. We utilises pGEX-6P-1 to express this parts to get nsp5 in its native state and verified its activity (Km/kcat=27,691 s⁻¹M⁻¹) through FRET. This part together with BBa_K5131008 can be used as an in vivo inhibitor screening platform for nsp5.

Sequence design of pGEX-GST-nsp5_nativte-His

In SARS-CoV-2 lifecycle, nsp5 must undergo self-cleavage at the N-terminus to release itself from the polyprotein[1]. After learning from BBa_K5131000, we introduced four amino acids (AVLQ) at the N-terminus of nsp5 to facilitate its self-cleavage from the GST tag, resulting in naive N- and C-termini. The rest of the sequence remained consistent with BBa_K5131000 . (Figure 1).

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

Construction of pGEX-GST-nsp5_native-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_native-His. B) PCR amplification of nsp5_native and pGEX-6P-1. C) Sequencing validation of nsp5_native.

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-termini.

Figure 3. Purification of nsp5. Lane 1-3: marker, 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, respectively. Finally, 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 .

Reference:
1. Yang H, Rao Z. Structural biology of SARS-CoV-2 and implications for therapeutic development. Nat Rev Microbiol. 2021 Nov;19(11):685-700. doi: 10.1038/s41579-021-00630-8. Epub 2021 Sep 17. PMID: 34535791; PMCID: PMC8447893. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 402