Difference between revisions of "Part:BBa K5131000"
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Our project aimes to construct an in vivo SARS-CoV-2 nsp5 inhibitor screening platform, so we first needed to verify that the nsp5 we expressed in <html><i>E. coli</I></html> BL21 was functional. We utilises pGEX-6P-1 to express this part(<bbpart>BBa_K5131006</bbpart>). However, the molecular weight of the purified protein differs from the expected molecular weight of nsp5 (33.8 kDa). We determined the reason and investigated how the GST tag might affect nsp5 enzymatic activity based on structure prediction.
 
Our project aimes to construct an in vivo SARS-CoV-2 nsp5 inhibitor screening platform, so we first needed to verify that the nsp5 we expressed in <html><i>E. coli</I></html> BL21 was functional. We utilises pGEX-6P-1 to express this part(<bbpart>BBa_K5131006</bbpart>). However, the molecular weight of the purified protein differs from the expected molecular weight of nsp5 (33.8 kDa). We determined the reason and investigated how the GST tag might affect nsp5 enzymatic activity based on structure prediction.
 
<h2> <b> About SARS-CoV-2 nsp5 </b> </h2>
 
<h2> <b> About SARS-CoV-2 nsp5 </b> </h2>
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Non-structural protein 5 (nsp5) is the most highly conserved non-structural protein across the Coronaviridae family, with a molecular weight of approximately 33 kDa[1]. In SARS-CoV-2, it's referred to as the main protease due to its pivotal role in the cleavage of polyproteins. Nsp5 has up to 11 conserved cleavage sites on the polyprotein and first undergoes autolytic cleavage to release itself, after which it cleaves at these sites to release other non-structural proteins.
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Non-structural protein 5 (nsp5) is the most highly conserved non-structural protein across the Coronaviridae family, with a molecular weight of approximately 33 kDa<sup>[1]</sup>. In SARS-CoV-2, it's referred to as the main protease due to its pivotal role in the cleavage of polyproteins. Nsp5 has up to 11 conserved cleavage sites on the polyprotein and first undergoes autolytic cleavage to release itself, after which it cleaves at these sites to release other non-structural proteins.
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Nsp5 consists of three structural domains: two N-terminal domains with protease activity and a C-terminal domain composed of &#945;-helices. The active form of SARS-CoV-2 nsp5 is a homodimer that recognizes substrates approximately 10 amino acid residues in length, but exhibits selectivity at only four specific positions[2]. In addition to cleaving viral proteins, nsp5 also suppresses the host innate immune response by degrading host protein factors[3]. In this project, our primary goal is to express functional nsp5 in E.coli BL21 in preparation for in vivo inhibitor screening. Additionally, since nsp5 has the ability to recognize and cleave specific sequences, we also aim to develop it as a tool enzyme for removing recombinant tags during protein purification.
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Nsp5 consists of three structural domains: two N-terminal domains with protease activity and a C-terminal domain composed of &#945;-helices. The active form of SARS-CoV-2 nsp5 is a homodimer that recognizes substrates approximately 10 amino acid residues in length, but exhibits selectivity at only four specific positions<sup>[2]</sup>. In addition to cleaving viral proteins, nsp5 also suppresses the host innate immune response by degrading host protein factors<sup>[3]</sup>. In this project, our primary goal is to express functional nsp5 in <i>E. coli</i> BL21 in preparation for in vivo inhibitor screening. Additionally, since nsp5 has the ability to recognize and cleave specific sequences, we also aim to develop it as a tool enzyme for removing recombinant tags during protein purification.
 
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<h2> <b> Express validation of nsp5 </b> </h2>
 
<h2> <b> Express validation of nsp5 </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 analysis showed that the purified and cleaved protein had high purity. However, the band appeared around 70 kDa(Figure 3), which differs from the expected molecular weight of nsp5 (33.8 kDa). Purification details can be seen in <html><a href="https://2024.igem.wiki/squirrel-shanghai/engineering">Engineering Success</a></html>
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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 analysis showed that the purified and cleaved protein had high purity. However, the band appeared around 70 kDa(Figure 3), which differs from the expected molecular weight of nsp5 (33.8 kDa). Purification details can be seen in <html><a href="https://2024.igem.wiki/squirrel-shanghai/engineering">Engineering Success</a></html>
 
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<div style="text-align:center;">
 
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Revision as of 13:26, 2 October 2024


SARA-Cov-2 nsp5

Our project aimes to construct an in vivo SARS-CoV-2 nsp5 inhibitor screening platform, so we first needed to verify that the nsp5 we expressed in E. coli BL21 was functional. We utilises pGEX-6P-1 to express this part(BBa_K5131006). However, the molecular weight of the purified protein differs from the expected molecular weight of nsp5 (33.8 kDa). We determined the reason and investigated how the GST tag might affect nsp5 enzymatic activity based on structure prediction.

About SARS-CoV-2 nsp5

Non-structural protein 5 (nsp5) is the most highly conserved non-structural protein across the Coronaviridae family, with a molecular weight of approximately 33 kDa[1]. In SARS-CoV-2, it's referred to as the main protease due to its pivotal role in the cleavage of polyproteins. Nsp5 has up to 11 conserved cleavage sites on the polyprotein and first undergoes autolytic cleavage to release itself, after which it cleaves at these sites to release other non-structural proteins. Nsp5 consists of three structural domains: two N-terminal domains with protease activity and a C-terminal domain composed of α-helices. The active form of SARS-CoV-2 nsp5 is a homodimer that recognizes substrates approximately 10 amino acid residues in length, but exhibits selectivity at only four specific positions[2]. In addition to cleaving viral proteins, nsp5 also suppresses the host innate immune response by degrading host protein factors[3]. In this project, our primary goal is to express functional nsp5 in E. coli BL21 in preparation for in vivo inhibitor screening. Additionally, since nsp5 has the ability to recognize and cleave specific sequences, we also aim to develop it as a tool enzyme for removing recombinant tags during protein purification.

Figure 1. Cleavage site of nsp5 and its structure[2].

Sequence design of pGEX-GST-nsp5-His

To ensure soluble expression of the protein, we selected the pGEX-6P-1 vector, which includes a GST tag and an HRV 3C protease cleavage site. For purification, we fused a 6*His tag to the C-terminus of the nsp5 sequence while retaining the HRV 3C protease cleavage site for subsequent removal of the tag(Figure 1).

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

Construction of pGEX-GST-nsp5-His

We first successfully amplified the vector backbone and the nsp5-6His tag separately using PCR (Figure 2B). Subsequently, we constructed the pGEX-GST-nsp5-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 of nsp5

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 analysis showed that the purified and cleaved protein had high purity. However, the band appeared around 70 kDa(Figure 3), which differs from the expected molecular weight of nsp5 (33.8 kDa). Purification details can be seen in Engineering Success

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
After carefully reviewing the clone, we discovered that the theoretical molecular weight of the GST tag + nsp5 was approximately 60 kDa, which was close to the observed band position. This indicated that the larger-than-expected molecular weight was likely due to the incomplete removal of the GST tag. Since there were no redundant amino acids between the HRV 3C protease cleavage site and the nsp5-GST tag, we hypothesized that steric hindrance at the cleavage site was preventing HRV 3C protease from accessing the site and cleaving the GST tag. To validate our hypothesis., we used AlphaFold3 to predict the structure of GST-nsp5 (Figure 4). The results revealed that GST-nsp5 forms a centrosymmetric dimer, and upon closely examining the HRV 3C protease recognition region (shown in cyan), we observed that this sequence folds inward into the nsp5 structure, with the cleavage site residues 226Q and 227G positioned at the deepest part of the nsp5 cavity. This suggests that nsp5 directly blocks HRV 3C protease from accessing the cleavage site, thereby preventing the removal of the N-terminal GST tag.
Figure 4. Predicted structure of GST-nsp5, where nsp5 is shown in orange, the GST tag in green, the HRV 3C protease recognition region in cyan, and the amino acid residues at the cleavage site in yellow.
We also investigated whether the presence of the GST tag might affect nsp5 enzymatic activity. To do so, we compared the structures of GST-nsp5 and the nsp5-substrate complex (PDB: 7DVP) (Figure 5). Interestingly, we found that the HRV 3C protease recognition region (shown in cyan) overlaps with the site where nsp5 recognizes its natural substrate (shown in violet). This indicates that the presence of the GST tag and the HRV 3C protease recognition sequence may interfere with substrate recognition by nsp5. This prompted us to further design a new composite part, pGEX-GST-nsp5_native-His(BBa_K5131007), to express nsp5 with a native N- and C-terminus.
Figure 5. Structural comparison of GST-nsp5 and the nsp5-substrate complex (PDB: 7DVP), where nsp5 is shown in orange, the GST tag in green, the HRV 3C protease recognition region in cyan, nsp5 (PDB: 7DVP) in red, and the substrate (PDB: 7DVP) in violet.


Reference:
1. Jin Z, Du X, Xu Y, Deng Y, Liu M, Zhao Y, Zhang B, Li X, Zhang L, Peng C, Duan Y, Yu J, Wang L, Yang K, Liu F, Jiang R, Yang X, You T, Liu X, Yang X, Bai F, Liu H, Liu X, Guddat LW, Xu W, Xiao G, Qin C, Shi Z, Jiang H, Rao Z, Yang H. Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors. Nature. 2020 Jun;582(7811):289-293. doi: 10.1038/s41586-020-2223-y. Epub 2020 Apr 9. PMID: 32272481.

2. Zhao Y, Zhu Y, Liu X, Jin Z, Duan Y, Zhang Q, Wu C, Feng L, Du X, Zhao J, Shao M, Zhang B, Yang X, Wu L, Ji X, Guddat LW, Yang K, Rao Z, Yang H. Structural basis for replicase polyprotein cleavage and substrate specificity of main protease from SARS-CoV-2. Proc Natl Acad Sci U S A. 2022 Apr 19;119(16):e2117142119. doi: 10.1073/pnas.2117142119. Epub 2022 Apr 5. PMID: 35380892; PMCID: PMC9172370.

3.Wu Y, Ma L, Zhuang Z, Cai S, Zhao Z, Zhou L, Zhang J, Wang PH, Zhao J, Cui J. Main protease of SARS-CoV-2 serves as a bifunctional molecule in restricting type I interferon antiviral signaling. Signal Transduct Target Ther. 2020 Oct 6;5(1):221. doi: 10.1038/s41392-020-00332-2. PMID: 33024073; PMCID: PMC7537955.


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 390