Part:BBa_K5321010

GFP_truncated_linker(TEV)_YFP_truncated

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 148
  Illegal AgeI site found at 159
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

We used MBP_linker(TEV)_GFP_truncated_linker(PPV)_YFP_truncated as a cleavage substrate for Tobacco Etch Virus protease (TEVp), which is formed by a TEVp cleavage site (ENLYFQS) between MBP soluble tag and truncated Green fluorescent protein (GFP)-truncated Yellow fluorescent protein (YFP) fusion protein. When it is cleaved by TEVp, two bands of different sizes can be observed by SDS-PAGE electrophoresis.Also, there is a PPVp leavage site between the truncated GFP and YFP, which can be used to test the activity of PPVp as well.

Figure 1 | Schematic of MBP_linker(TEV)_GFP_truncated_linker(PPV)_YFP_truncated.

Characterization

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 intact 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 2 | 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.