Part:BBa_K5321007

N-terminal of plum pox virus protease (nPPVp), codon optimized for E. coli, 6x His and FRB tagged

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Illegal PstI site found at 649
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Illegal BglII site found at 291
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COMPATIBLE WITH RFC[1000]

Usage and Biology

Split plum pox virus protease (PPVp), also referred to as the nuclear inclusion a protein (NIa), is a key enzyme from the plum pox virus (PPV). It plays a critical role as one of the three viral proteases involved in the maturation of the viral polyprotein into its functional components. Here, we used it as a tool to amplify our detection signal of disease biomarkers.

This part is the coding sequence for N-terminal fragment (nPPVp). 6x His tag was added to the front of the peptide for purification. FRB is a tag in mTOR signaling pathway. It could link with Rapamycin and in cascade form a FKBP-RAP-FRB complex, causing the split protease to heterodimerize and form a functional protease.

Figure 1 | Prediction result of PPVp structure. Cyan portion represents the N-terminal fragment (nPPVp), while green portion represents the C-terminal fragment (cPPVp).

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 | Elution profile of nPPV. Peak A stands for transmission peak; peak B stands for elution peak of nPPV.

Figure 3 | SDS-PAGE analysis of the cell lysate, the transmission peak and the elution peak after nickel affinity chromatography through AKTA or gravity chromatography. Lane2-4, total proteins of bacteria expressing cPPV, elution peak, transmission peak of cPPV; Lane5-7, total proteins of bacteria expressing nPPV; Lane9-10, elution peak and transmission peak of nPPV. All the proteins are purified through AKTA. Because of the low resolution of the picture, other bands of low expression proteins can’t be seen.

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 PPV 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. For the split PPV protease, we additionally added a fixed amount of rapamycin to induce protease dimerization and activation.

Figure 4 | SDS-PAGE analysis of protease activity verification in split and full-length PPV constructs. Lane M: Marker; Lane Split-2: Split PPVp with 700 nM rapamycin at 10, 30, and 120 min; Lane Split-3: Split PPVp with 1400 nM rapamycin at 10, 30, and 120 min; Lane PPV: Full-length PPVp with its substrate at 10, 30, and 120 min.

The results show that the split PPVp exhibited protease activity, as evidenced by the reduction in substrate over time.

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 | Solubility analysis of nPPV. SU: supernatant after ultrasonication; PU: precipitate after ultrasonication. The figure shows solubility analysis of the two split enzymes. MW: FRB-nPPV: 24.9kDa; FKBP-cPPV: 27.3kDa. The result shows that almost all of the FRB-nPPV and FKBP-cPPV exist in PU but not SU, which indicates the insoluble state(inclusion body) of the proteins. In contrast, the EGFP control mainly appears in the soluble component(SU), proving the correctness of our protocol. The unlabeled electrophoresis bands are due to sample loading mistakes.

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