Difference between revisions of "Part:BBa K5131006"

Revision as of 06:56, 1 October 2024

pGEX-GST-nsp5-His

Information of SARS-Cov-2 nsp5 can be seen in BBa_K5131000. We use this part to express and purify SARS-Cov-2 nsp5. 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.

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_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

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 kD(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_native. Lane 1-3: marker, 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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1726
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 770
Illegal XhoI site found at 1721
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1165
Illegal SapI.rc site found at 167

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-	<img src="https://static.igem.wiki/teams/5131/engineering/engineering-success-1.webp" style="width: 80%;">
+	<img src="https://static.igem.wiki/teams/5131/engineering/engineering-success-5.webp" style="width: 80%;">
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-			<b>Figure 1. </b> Sequence design for GST-nsp5-His. ”↓“for the cleavage site.
+			<b>Figure 1. </b> Sequence design for GST-nsp5_native-His. ”↓“for the cleavage site.
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 <h2> <b> Construction of pGEX-GST-nsp5-His </b> </h2>
-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).
+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).
 <html>
 <div style="text-align:center;">
-	<img src="https://static.igem.wiki/teams/5131/engineering/engineering-success-13.webp" style="width: 75%;">
+	<img src="https://static.igem.wiki/teams/5131/engineering/engineering-success-6.webp" style="width: 75%;">
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 		<caption>
-			<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.
+			<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.
 	</div>
 </div>
 </html>
 <h2> <b> Express validation nsp5 </b> </h2>
-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 kD(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>
+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 kD(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>
 <html>
 <div style="text-align:center;">
-	<img src="https://static.igem.wiki/teams/5131/engineering/engineering-success-3.webp" style="width: 75%;">
+	<img src="https://static.igem.wiki/teams/5131/engineering/engineering-success-7.webp" style="width: 75%;">
 	<div style="text-align:center;">
 		<caption>
-			<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
+			<b>Figure 3. </b>Purification of nsp5_native. Lane 1-3: marker, digested nickel beads, purified protein.
 	</div>
 </div>