Difference between revisions of "Part:BBa K5321005"

 
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===Sequence and Features===
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===Usage and Biology===
  
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TEV protease (Tobacco Etch Virus nuclear-inclusion-a endopeptidase) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV).The tobacco etch virus encodes its entire genome as a single massive polyprotein (350 kDa). This is cleaved into functional units by the three proteases: P1 protease (1 cleavage site), helper-component protease (1 cleavage site) and TEV protease (7 cleavage sites). We used it to transform the protein signal to a visible output of hCG.
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'''Figure 1 | Structure of TEVp.''' The two beta-barrels are its functional domain.<br>
  
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===Characterization===
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===Usage and Biology===
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====Protein Purification====
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Because our system is an in vitro detection system, it’s essential for us to express proteins and then purify them. We have chosen three strategies to express proteins. Directly expression with 6xHis tag but without solubility tags, expression with both 6xHis tag and solubility tags and expression with cell-free system. Methodology of ours for purification is affinity chromatography. To be specific, we use nickel affinity chromatography to purify proteins with 6xhis tag (with or without solubility tags), and use glutathione affinity chromatography to purify proteins expressed by cell-free system. For nickel affinity chromatography, we use both ÄKTA system and gravity chromatography. For glutathione affinity chromatography, we use ÄKTA system. Furthermore, for proteins that are not expressed with solubility tags or cell-free system, they usually form inclusion bodies and we need new strategy to tackle this tricky problem. On-column refolding is the very solution applied by us. Finally, to test whether the affinity chromatography works as expected, we have done SDS-PAGE analysis to see the purification results.
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'''Figure 2 | SDS-PAGE analysis of  the elution peak after gravity chromatography.''' Lane1-3, elution peak after gravity chromatography. The results demonstrated that TEV was successfully purified.
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====Protease Activity Verification====
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Since our system relies on the protease both amplifying the signal and triggering the release of the final colloidal gold output, it is crucial to verify the target protease activity to ensure that the enzymes used in our experiments are active and functioning as expected. To achieve this, we designed a experiment to verify the enzyme activity under controlled conditions (you can find more detailed information about this experiment in our protocol). We validated the activity of two intact proteases and one split protease. For the TEV proteases, we mixed a calculated amount of the enzyme with its corresponding substrate and added the appropriate amount of reaction buffer. The mixture was incubated at 30°C, and samples were taken at different time points. The reaction was stopped with SDS loading buffer, followed by electrophoresis. Enzyme activity was confirmed by observing the reduction in substrate and the presence of cleavage product bands.
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'''Figure 3 | Enzymatic activity assay of TEV protease extracted from inclusion bodies.''' The figure shows the SDS-PAGE analysis of the enzymatic activity of TEV protease on its substrate, under two different reaction buffer conditions (Tris-HCl: 20 mM Tris-HCI+10 mM NaCl+10 mM KCI+1 mM DTT and HEPES: 20 mM HEPES+10 mM NaCI+10 mM KCI+1 mM DTT). The top band corresponds to the intact substrate (tGFP-tevS-tYFP), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products (tGFP and tYFP), which increase over time. Samples were taken at 0, 5, 30, 120, and 240 minutes, with the reaction stopped by adding SDS loading buffer and heating at 95°C for 5-10 minutes.The results confirm that the TEV protease extracted from inclusion bodies is active.
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====Protein Solubility Analysis====
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We further examined the solubility of our target proteins. It is achieved by 3s ultrasonication on ice + 10s interval, power 300W, for 40 minutes to completely destruct bacterial structure. Then the sample is centrifuged. The soluble and insoluble components will appear in the supernatant and the precipitate respectively. With 5×SDS loading buffer treated, the two parts can be used for downstream SDS-PAGE analysis. As a control, EGFP, which is soluble in E.coli, is also expressed and analyzed with the same protocol.
  
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'''Figure 5 | SDS-PAGE analysis of protein solubility of TEV.''' AU: after ultrasonication; SU: supernatant after ultrasonication; FS: final supernatant. Lane1, AU 1-1, i.e., total proteins of bacteria after ultrasonication; Lane2, SU1-1, i.e., proteins in the supernatant after ultrasonication; Lane3, FS1-1, i.e., proteins in the final supernatant. MW of TEV is around 31.0 KDa. This result demonstrated that bacteria expressed TEV successfully and the TEV was mostly in the final supernatant.
  
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===References===
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===Functional Parameters===
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1. Fink, T., Lonzarić, J., Praznik, A., Plaper, T., Merljak, E., Leben, K., Jerala, N., Lebar, T., Strmšek, Ž., Lapenta, F., Benčina, M., & Jerala, R. (2019). Design of fast proteolysis-based signaling and logic circuits in mammalian cells. ''Nature chemical biology'', ''15''(2), 115–122. https://doi.org/10.1038/s41589-018-0181-6<br>
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Revision as of 11:13, 22 September 2024

Plum pox virus protease (PPVp), codon optimized for E. coli, 6x His tagged

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 712
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 712
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 712
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 712
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

TEV protease (Tobacco Etch Virus nuclear-inclusion-a endopeptidase) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV).The tobacco etch virus encodes its entire genome as a single massive polyprotein (350 kDa). This is cleaved into functional units by the three proteases: P1 protease (1 cleavage site), helper-component protease (1 cleavage site) and TEV protease (7 cleavage sites). We used it to transform the protein signal to a visible output of hCG.


Figure 1 | Structure of TEVp. The two beta-barrels are its functional domain.

Characterization

Protein Purification

Because our system is an in vitro detection system, it’s essential for us to express proteins and then purify them. We have chosen three strategies to express proteins. Directly expression with 6xHis tag but without solubility tags, expression with both 6xHis tag and solubility tags and expression with cell-free system. Methodology of ours for purification is affinity chromatography. To be specific, we use nickel affinity chromatography to purify proteins with 6xhis tag (with or without solubility tags), and use glutathione affinity chromatography to purify proteins expressed by cell-free system. For nickel affinity chromatography, we use both ÄKTA system and gravity chromatography. For glutathione affinity chromatography, we use ÄKTA system. Furthermore, for proteins that are not expressed with solubility tags or cell-free system, they usually form inclusion bodies and we need new strategy to tackle this tricky problem. On-column refolding is the very solution applied by us. Finally, to test whether the affinity chromatography works as expected, we have done SDS-PAGE analysis to see the purification results.


Figure 2 | SDS-PAGE analysis of the elution peak after gravity chromatography. Lane1-3, elution peak after gravity chromatography. The results demonstrated that TEV was successfully purified.

Protease Activity Verification

Since our system relies on the protease both amplifying the signal and triggering the release of the final colloidal gold output, it is crucial to verify the target protease activity to ensure that the enzymes used in our experiments are active and functioning as expected. To achieve this, we designed a experiment to verify the enzyme activity under controlled conditions (you can find more detailed information about this experiment in our protocol). We validated the activity of two intact proteases and one split protease. For the TEV proteases, we mixed a calculated amount of the enzyme with its corresponding substrate and added the appropriate amount of reaction buffer. The mixture was incubated at 30°C, and samples were taken at different time points. The reaction was stopped with SDS loading buffer, followed by electrophoresis. Enzyme activity was confirmed by observing the reduction in substrate and the presence of cleavage product bands.

Figure 3 | Enzymatic activity assay of TEV protease extracted from inclusion bodies. The figure shows the SDS-PAGE analysis of the enzymatic activity of TEV protease on its substrate, under two different reaction buffer conditions (Tris-HCl: 20 mM Tris-HCI+10 mM NaCl+10 mM KCI+1 mM DTT and HEPES: 20 mM HEPES+10 mM NaCI+10 mM KCI+1 mM DTT). The top band corresponds to the intact substrate (tGFP-tevS-tYFP), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products (tGFP and tYFP), which increase over time. Samples were taken at 0, 5, 30, 120, and 240 minutes, with the reaction stopped by adding SDS loading buffer and heating at 95°C for 5-10 minutes.The results confirm that the TEV protease extracted from inclusion bodies is active.


Protein Solubility Analysis

We further examined the solubility of our target proteins. It is achieved by 3s ultrasonication on ice + 10s interval, power 300W, for 40 minutes to completely destruct bacterial structure. Then the sample is centrifuged. The soluble and insoluble components will appear in the supernatant and the precipitate respectively. With 5×SDS loading buffer treated, the two parts can be used for downstream SDS-PAGE analysis. As a control, EGFP, which is soluble in E.coli, is also expressed and analyzed with the same protocol.

Figure 5 | SDS-PAGE analysis of protein solubility of TEV. AU: after ultrasonication; SU: supernatant after ultrasonication; FS: final supernatant. Lane1, AU 1-1, i.e., total proteins of bacteria after ultrasonication; Lane2, SU1-1, i.e., proteins in the supernatant after ultrasonication; Lane3, FS1-1, i.e., proteins in the final supernatant. MW of TEV is around 31.0 KDa. This result demonstrated that bacteria expressed TEV successfully and the TEV was mostly in the final supernatant.

References

1. Fink, T., Lonzarić, J., Praznik, A., Plaper, T., Merljak, E., Leben, K., Jerala, N., Lebar, T., Strmšek, Ž., Lapenta, F., Benčina, M., & Jerala, R. (2019). Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nature chemical biology, 15(2), 115–122. https://doi.org/10.1038/s41589-018-0181-6