Difference between revisions of "Part:BBa K316012"
| Line 88: | Line 88: | ||
===Documentation=== | ===Documentation=== | ||
| + | The '''Tobacco etch virus (TEV) protease''' is a 27 kDa catalytic domain of the nuclear inclusion polyprotein a (NIa) in TEV. It specifically recognizes the amino acid sequence ENLYFQG/S and cleaves between the Q and G/S residues. TEV belongs to the ''Potyviridae'' family of viruses, which includes other positive-strand RNA viruses. The TEV genome is translated into a single large polyprotein that is subsequently processed by virus-encoded proteins with proteolytic activity. Despite its substrate specificity, the use of TEV protease is limited due to its self-inactivation through autocleavage and its low solubility during purification, caused by its high hydrophobicity <ref>Reference 1</ref>. | ||
| + | Studies have shown that TEV protease undergoes self-cleavage near its C terminus after residue 218, at a site that does not follow the canonical sequence, resulting in a truncated protein with reduced activity <ref>Reference 2</ref>. According to UniProt, its active sites are in 214, 223, 256, 649, 722, 2083, 2118, and 2188 <ref>Reference 3</ref>. | ||
| + | |||
| + | '''In this study, the sequences of mutant TEV protease S219P and wild type 1LVM were compared to determine if TEV S219P exhibits greater efficiency and interaction with a substrate.''' Structure-based sequence alignment was performed using ESPript3, and both enzymes were modeled with AlphaFold for structural analysis. Additionally, heatmaps of protein stability were generated using Protein-Sol, and molecular docking was carried out with HADDOCK 2.4 and BIOVIA Discovery Studio Visualizer to compare their interactions. | ||
| + | |||
| + | Initially, the sequences of TEV protease S219P and TEV 1LVM were analyzed using Clustal Omega to generate a protein alignment in ALN format, which was subsequently processed with ESPript3. ESPript3 provides a detailed representation of sequence similarities and secondary structure information for the aligned sequences. As shown in '''Figure 1''', both sequences exhibit a high degree of similarity. However, the TEV S219P sequence consists of 237 amino acids, while the TEV 1LVM sequence contains 229 amino acids, indicating that TEV S219P is longer. | ||
| + | |||
| + | In panel '''A''', differences are observed between amino acids 1-8 of TEV 1LVM and TEV S219P, attributed to the presence of a His-tag, as well as at position 10. Additionally, amino acids 229-237 in TEV S219P represent seven extra residues not present in TEV 1LVM. The differing amino acids between the two proteases are highlighted in yellow. The catalytic sites for both proteases are located at amino acids 214 and 223, as indicated by green triangles in panel A of '''Figure 1'''. The peptidase C4 domain, marked in dark blue, spans residues 1-218, corresponding to the coding sequence shared by both proteins. | ||
| + | |||
| + | In panel '''B''', the domains of chain A of TEV 1LVM, selected for comparison with TEV S219P, are illustrated. The domains of 1lvmA01 are shown in aqua, while those of 1lvmA02 are highlighted in pink. Table 1 in '''Figure 1''' provides a comprehensive description of these domains. | ||
| Line 94: | Line 104: | ||
<center> | <center> | ||
<figure> | <figure> | ||
| − | <img src='https://static.igem.wiki/teams/5150/parts/ | + | <img src='https://static.igem.wiki/teams/5150/parts/prote-n-alignment-of-tev-1lvm-with-tev-s219p-and-characteristics-of-tev-1lvm-domains-in-chai.jpg' width='500px' height='500px' /> |
| − | + | <figcaption> | |
| − | + | <b><small><i>Figure 1. Protein alignment of TEV 1LVM with TEV S219P and characteristics of TEV 1LVM domains in chain A. Image A retrieved from ESPript.</i></small></b> | |
| − | </figure></center></html | + | </figcaption> |
| + | </figure> | ||
| + | </center> | ||
| + | </html> | ||
Revision as of 22:02, 29 September 2024
TEV protease S219P autocatalysis resistant variant
TEV protease S219P autocatalysis resistant variant. This part had been reversed for the 3' strand in order to reduce any read-through that may be caused by upstream elements.
Introduction :
This is the nuclear inclusion protease, endogenous to Tobacco Etch Virus and is used in the late lifecycle to cleave polyprotein precursors. The recognition sequence is ENLYFQG/S 1 between QG or QSDue to it’s stringent sequence specificity, TEV is commonly used to cleave genetically engineered proteins.
Uses:
TEV proteinase is used to cleave fusion proteins. It is useful due to its high degree of specificity1 and potential to be used in vivo or in vitro applications.
Auto-inactivation:
Wild type TEV protease also cleaves itself at Met 218 and Ser 2192. This leads to auto-inactivation of the TEV protease and progressive loss of activity of the protein. The rate of inactivation is proportional to the concentration of protease. More stable Mutants have been produced by single amino acid substitutions S219V (AGC(serine) to GTG(valine) and S219P (AGC(serine) to CCG(proline)3.
Table I.
Kinetic parameters for wild-type and mutant TEV proteases with the peptide substrate TENLYFQSGTRR-NH2. From original paper by Kapust et.al. 20013
| Enzyme | Km (mM) | kcat (s-1) | kcat /Km (mM-1 s-1) |
| Wild type | 0.061 ± 0.010 | 0.16 ± 0.01 | 2.62 ± 0.46 |
| S219V | 0.041± 0.010 | 0.19 ± 0.01 | 4.63 ± 1.16 |
| S219P | 0.066 ± 0.008 | 0.09 ±0.01 | 1.36 ± 0.22 |
S219V* - retains same activity as wild type
S219P* - virtually imperivious to autocatalysis
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 32
- 1000COMPATIBLE WITH RFC[1000]
References
<biblio>
- 1 pmid=8179197
- 2 pmid=7793070
- 3 pmid=2047602
</biblio>
For more information about our project please visit our wiki.
Contribution
- Group: TecCEM, iGEM 2024
- Authors: Giovana Andrea Osorio León, Ana Laura Torres Huerta, Aurora Antonio Pérez, Lorena Gallegos Solís
- Summary: The sequences of TEV proteases S219P and 1LVM were compared to determine if TEV S219P exhibits greater efficiency and interaction with a substrate. Sequence alignment was performed using ESPript3, and both enzymes were modeled with AlphaFold for structural analysis. Additionally, heatmaps of protein stability were generated using Protein-Sol and molecular docking was carried out with HADDOCK 2.4 and BIOVIA Discovery Studio Visualizer to compare their interactions.
Documentation
The Tobacco etch virus (TEV) protease is a 27 kDa catalytic domain of the nuclear inclusion polyprotein a (NIa) in TEV. It specifically recognizes the amino acid sequence ENLYFQG/S and cleaves between the Q and G/S residues. TEV belongs to the Potyviridae family of viruses, which includes other positive-strand RNA viruses. The TEV genome is translated into a single large polyprotein that is subsequently processed by virus-encoded proteins with proteolytic activity. Despite its substrate specificity, the use of TEV protease is limited due to its self-inactivation through autocleavage and its low solubility during purification, caused by its high hydrophobicity [1].
Studies have shown that TEV protease undergoes self-cleavage near its C terminus after residue 218, at a site that does not follow the canonical sequence, resulting in a truncated protein with reduced activity [2]. According to UniProt, its active sites are in 214, 223, 256, 649, 722, 2083, 2118, and 2188 [3].
In this study, the sequences of mutant TEV protease S219P and wild type 1LVM were compared to determine if TEV S219P exhibits greater efficiency and interaction with a substrate. Structure-based sequence alignment was performed using ESPript3, and both enzymes were modeled with AlphaFold for structural analysis. Additionally, heatmaps of protein stability were generated using Protein-Sol, and molecular docking was carried out with HADDOCK 2.4 and BIOVIA Discovery Studio Visualizer to compare their interactions.
Initially, the sequences of TEV protease S219P and TEV 1LVM were analyzed using Clustal Omega to generate a protein alignment in ALN format, which was subsequently processed with ESPript3. ESPript3 provides a detailed representation of sequence similarities and secondary structure information for the aligned sequences. As shown in Figure 1, both sequences exhibit a high degree of similarity. However, the TEV S219P sequence consists of 237 amino acids, while the TEV 1LVM sequence contains 229 amino acids, indicating that TEV S219P is longer.
In panel A, differences are observed between amino acids 1-8 of TEV 1LVM and TEV S219P, attributed to the presence of a His-tag, as well as at position 10. Additionally, amino acids 229-237 in TEV S219P represent seven extra residues not present in TEV 1LVM. The differing amino acids between the two proteases are highlighted in yellow. The catalytic sites for both proteases are located at amino acids 214 and 223, as indicated by green triangles in panel A of Figure 1. The peptidase C4 domain, marked in dark blue, spans residues 1-218, corresponding to the coding sequence shared by both proteins.
In panel B, the domains of chain A of TEV 1LVM, selected for comparison with TEV S219P, are illustrated. The domains of 1lvmA01 are shown in aqua, while those of 1lvmA02 are highlighted in pink. Table 1 in Figure 1 provides a comprehensive description of these domains.
