Difference between revisions of "Part:BBa K5321009"

Latest revision as of 13:32, 2 October 2024

GFP_truncated_linker(PPV)_YFP_truncated

Sequence and Features

Usage and Biology

We used GFP_truncated_linker(PPV)_YFP_truncated as a cleavage substrate for Plum pox virus protease (PPVp), which is formed by a PPVp cleavage site (NVVVHQA) with truncated Green fluorescent protein (GFP) and the truncated Yellow fluorescent protein (YFP) at both ends, and there is a linker between each of the two, consisting of G and S. When it is cleaved by PPVp, two bands of different sizes can be observed by SDS-PAGE electrophoresis.

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 GFP_truncated_linker(PPV)_YFP_truncated. Peak A stands for transmission peak, peak B stands for elution peak of GFP_truncated_linker(PPV)_YFP_truncated.

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, total proteins of bacteria expressing GFP_truncated_linker(PPV)_YFP_truncated; Lane3, elution peak of GFP_truncated_linker(PPV)_YFP_truncated through AKTA; Lane4, total proteins of bacteria expressing PPV; Lane5, elution peak of PPV through AKTA; Lane6, elution peak of GFP_truncated_linker(PPV)_YFP_truncated through gravity chromatography; Lane7, elution peak of PPV through gravity chromatography. PPV wasn’t purified successfully in this picture.

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. (A)：Lane M: Marker; Lane 0: Split-1 at 0 min; Lane Split-1: Split PPVp with 350 nM rapamycin at 10, 30, and 120 min; Lane Split-N: Split PPVp without rapamycin at 10, 30, and 120 min; Lane cPPV: Full-length FKBP-cPPVp with 1400 nM rapamycin at 10, 30, and 120 min; Lane nPPV: Full-length FRB-nPPVp with 1400 nM rapamycin at 10, 30, and 120 min. (B)：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 both the full-length PPVp and the split PPVp (when induced by rapamycin) exhibit protease activity, as evidenced by the reduction in substrate over time. In contrast, a single split PPVp cannot cleave the substrate. However, when two split PPVs are combined without rapamycin induction, there is minimal cleavage activity, as indicated by the slight reduction in substrate from 10 to 30 and 120 minutes. The amount of substrate reduction is significantly less than that observed with rapamycin induction, leading to the conclusion that rapamycin is necessary for the optimal activity of the split PPVp.

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 GFP_truncated_linker(PPV)_YFP_truncated. SU: supernatant after ultrasonication; PU: precipitate after ultrasonication. MW: PPVp: 27.7kDa; GFP_truncated_linker(PPV)_YFP_truncated: 22.3kDa.

Both of the proteins are insoluble. The unlabeled electrophoresis bands are due to sample loading mistakes.