Difference between revisions of "Part:BBa K5131009"

 
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Information of SARS-Cov-2 nsp5 can be seen in <bbpart>BBa_K5131000</bbpart>. After characterization of <bbpart>BBa_K5131007</bbpart>, we found Km/kcat of nsp5 (27,691 s⁻¹M⁻¹) is much higher than TEV protease((Km/kcat =2,600 s⁻¹M⁻¹)) and HRV 3C protease(Km/kcat =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 Km/kcat 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.
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<h2> <b> Rational design of SARS-Cov-2 nsp5 </b> </h2>
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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.
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First, we predicted the structure of the wild-type nsp5 in complex with the linker substrate. Through structural analysis, we found that T21<html><sub>nsp5</sub></html> is distant from the catalytic center and interacts with only one amino acid of the substrate. Therefore, we chose to modify this site.
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To enhance the interaction between T211<html><sub>nsp5</sub></html>and the substrate, we aimed to replace T211<html><sub>nsp5</sub></html> 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 -CH<html><sub>2</sub></html>-CH<html><sub>3</sub></html>group, increasing the side chain's length.
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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<html><sub>substrate</sub></html>) exhibiting a rotation of approximately 50 degrees. Therefore, we focused on analyzing this region in detail (Figure 1).
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<img src="https://static.igem.wiki/teams/5131/model/model-8.webp" style="width: 75%;">
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<div style="text-align:center;">
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<caption>
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<b>Figure 1. </b>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.
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By comparing the structures, we found that when T21<html><sub>nsp5</sub></html> was replaced by I21<html><sub>nsp5</sub></html>, the R10<html><sub>substrate</sub></html> side chain, which previously interacted with T21<html><sub>nsp5</sub></html, was displaced from its original position. Although the interaction with residue 21 was weakened, this subtle conformational change allowed the carbonyl oxygen of G23<html><sub>nsp5</sub></html> to form additional interactions with the R10<html><sub>substrate</sub></html> side chain. Additionally, the distance between the carbonyl oxygens of T24<html><sub>nsp5</sub></html> and R10<html><sub>substrate</sub></html> decreased from 2.8 Å to 2.7 Å.
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In summary, in wild-type nsp5, only T24<html><sub>nsp5</sub></html> and T21<html><sub>nsp5</sub></html> interact with R10<html><sub>substrate</sub></html>, whereas in nsp5-T21I, residues I21<html><sub>nsp5</sub></html>, G23<html><sub>nsp5</sub></html>, and T24<html><sub>nsp5</sub></html> together stabilize R10<html><sub>substrate</sub></html>. We hypothesize that this change enhances the interaction between the substrate and nsp5, leading to improved enzymatic activity.
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<h2> <b> Construction of pGEX-GST-nsp5_T21I-His </b> </h2>
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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).
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<img src="https://static.igem.wiki/teams/5131/engineering/engineering-success-6.webp" style="width: 75%;">
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<div style="text-align:center;">
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<caption>
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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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<h2> <b> Express validation and characterization nsp5_T21I </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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<div style="text-align:center;">
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<img src="https://static.igem.wiki/teams/5131/engineering/engineering-success-7.webp" style="width: 75%;">
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<div style="text-align:center;">
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<caption>
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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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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 12:00, 1 October 2024


pGEX-GST-nsp5_T21I-His

Information of SARS-Cov-2 nsp5 can be seen in BBa_K5131000. After characterization of BBa_K5131007, we found Km/kcat of nsp5 (27,691 s⁻¹M⁻¹) is much higher than TEV protease((Km/kcat =2,600 s⁻¹M⁻¹)) and HRV 3C protease(Km/kcat =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 Km/kcat 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 T21nsp5 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 T211nsp5and the substrate, we aimed to replace T211nsp5 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(R10substrate) 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 T21nsp5 was replaced by I21nsp5, the R10substrate side chain, which previously interacted with T21nsp5nsp5 to form additional interactions with the R10substrate side chain. Additionally, the distance between the carbonyl oxygens of T24nsp5 and R10substrate decreased from 2.8 Å to 2.7 Å.

In summary, in wild-type nsp5, only T24nsp5 and T21nsp5 interact with R10substrate, whereas in nsp5-T21I, residues I21nsp5, G23nsp5, and T24nsp5 together stabilize R10substrate. 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 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 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_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


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