Part:BBa_K316012
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>
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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 its recognition site or synthetic substrate (ENLYFQG). 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.
To evaluate the impact of the differences in the sequence of the mutant with respect to the wild-type protein, modeling and simulation of the interaction with the target peptide were performed. Afterwards, the program ChimeraX was used to obtain the 3D model of both TEV proteases using the AlphaFold tool for structure prediction, as shown in Figure 2. The image labeled with letter A is the model obtained for the TEV protease S219P, and the image labeled with letter B is the model for chain A of TEV 1LVM. Both proteases were overlapped to compare them and observe similarities, as shown in image C. The overlap is highlighted in two colors: light blue for the TEV protease S219P and purple for TEV 1LVM, showing areas where the structures align or differ. It can be seen that most of the chains of both proteins align, as well as the main beta sheets and alpha helices, indicating that the S219P mutation does not significantly affect the native structure of the enzyme. However, there are regions at the ends where conformational differences between TEV S219P and TEV 1LVM are observed. These differences may influence the function or stability of the protein.
Subsequently, the Protein-Sol program was used to generate energy and charge heatmaps, providing insights into the stability of the proteases (TEV 1LVM and TEV S219P) under varying pH and ionic strength (salt concentration) conditions. The Debye-Hückel (DH) method was employed to model interactions between ionizable groups and to calculate pKa values. Each heatmap consists of 91 combinations of pH and ionic strength. For qualitative comparison with the experiment, the CH2 and CH3 domains of the IgG1 structure (PDB 1HZH) were used, as pH and ionic strength variations have been reported to affect the stability of these domains in IgG.[4]
TEV 1LVM
The heatmap results for TEV 1LVM are shown in Figure 3. The color scale ranges from red (positive values) to green (negative values). Positive values (ranging from red to orange) suggest conditions where the protease has higher energy, potentially indicating instability or less favorable folding. Conversely, negative values (green) indicate more stable conditions with lower energy.
At a pH between 2.0 and 4.0 and high ionic strengths (0.1–0.3 M), the TEV 1LVM protease displays higher energy values, which may indicate enzyme instability. As the pH increases beyond 5.0 and the ionic strength decreases, the energy values drop, suggesting that these conditions favor a more stable folded state of the protease. However, the optimal conditions for this enzyme are observed at a pH of 6.5 to 8.0 and low ionic strengths (0.005–0.1 M), which promote the stability of the TEV 1LVM protease[5]
- ↑ Nam, H., Hwang, B. J., Choi, D. Y., Shin, S., & Choi, M. (2020). Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self-cleavage exhibits enhanced enzymatic activity. FEBS Open Bio, 10(4), 619–626. https://doi.org/10.1002/2211-5463.12828
- ↑ Nunn, C. M., Jeeves, M., Cliff, M. J., Urquhart, G. T., George, R. R., Chao, L. H., Tsuchia, Y., & Djordjevic, S. (2005). Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site. Journal of Molecular Biology, 350(1), 145–155. https://doi.org/10.1016/j.jmb.2005.04.013
- ↑ P04517 · POLG_TEV. (2024). UniProt. https://www.uniprot.org/uniprotkb/P04517/entry
- ↑ [4]
- ↑ Hebditch, M., Warwicker, J. Web-based display of protein surface and pH-dependent properties for assessing the developability of biotherapeutics. Sci Rep 9, 1969 (2019). https://doi.org/10.1038/s41598-018-36950-8/entry
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