V8 Protease Purification
Plasmid Design & Construction
The V8 protease expression plasmid was modified to remove the signal peptide. A four-amino-acid mutation was introduced into the propeptide to prevent self-degradation of the V8 protease in the Escherichia coli system, facilitating easier protein purification. Using this modified plasmid, we successfully obtained the V8 protease precursor, which was later activated through enzymatic cleavage.
before:
LSSKAMDNHPQQTQSSKQQTPKIQKGGNLKPLEQREHAN
after:
LSSKAMDNHPQQTQSSKQQTPKIQKGGNLKPLQQRSHP
We synthesized the gene sequence of V8 protease according to the preferences of translation in Escherichia coli, using PCR to amplify both the pro and mature sequences of V8 protease while introducing four amino acid mutations. The constructed plasmid was confirmed by sequencing, and after amplification in DH5α, it was transformed into the BL21 (DE3) strain for expression and purification.
V8 Protease Expression and Purification
We synthesized the gene sequence of V8 protease, tailored to the codon preferences for efficient translation in Escherichia coli. Using PCR, we amplified both the pro and mature sequences of V8 protease, incorporating four specific amino acid mutations. The constructed plasmid was verified through sequencing, amplified in DH5α, and subsequently transformed into the BL21 (DE3) strain for expression and purification.
We added a His tag to the C-terminus of the constructed V8 protease and purified the protein using Nickel affinity chromatography. From the protein gel image, we observed the target protein at the sixth band in the marker (corresponding to a molecular weight of 39 kDa), which is consistent with the theoretical prediction. We also performed a Western blot using anti-His antibodies, and the results showed a positive signal, indicating that we successfully expressed the target protein.
Activation of V8 Protease
Next, we will use thermolysin to cleave the Ni-purified V8 protease protein for activation, resulting in V8 protease with the pro-sequence removed. After a second purification using a Q column, the gel image shows that the target protein band has shifted from the original sixth band to the fourth band, corresponding to a molecular weight of 25 kDa.
Enzyme Activity Assay
We used Z-leu-leu-glu-AMC as the substrate for V8 protease to test its activity. AMC (7-amino-4-methylcoumarin) is a fluorescent group commonly used in biochemistry for detecting protease activity. When cleaved by proteases, AMC releases a free amine that emits strong fluorescence upon excitation.After enzyme activation, we immediately incubated V8 protease with the substrate and promptly measured the fluorescence intensity of the fluorescent group using a microplate reader (Ex/Em: 340/460 nm) after adding the substrate.
Drug Screening
We aim to find a green and safe inhibitor of V8 protease. With this consideration, we have chosen several compound libraries for drug screening: the Natural Compound Library, Bioactive Compound Library, Clinical Compound Library, and Approved Drug Library. High-throughput screening technology uses microplates as experimental tools, allowing for convenient and rapid automatic liquid addition to quickly screen potential inhibitors of V8 protease. We have successfully purified active V8 protease, and we will further use a fluorescence system and a plate reader to measure fluorescence intensity as an indicator of V8 protease activity, combining this with high-throughput screening to identify potential inhibitors from the aforementioned compound libraries.
The V8 protein was diluted to final concentrations of 1 μM, 0.5 μM, 0.25 μM, and 0.125 μM, and the enzyme cleavage curves were measured using a microplate reader. The curves exhibited good linearity at concentrations greater than 0.125 μM, making them suitable for drug screening.
In our assay, we used 45 μL of V8 protein (final concentration 0.125 μM) and 5 μL of substrate (final concentration 20 μM), along with an inhibitor (10 μM) to dilute the protein. The substrate buffer consisted of enzyme activity test buffer at pH 8.0, containing 50 mM Tris-HCl, 5 mM EDTA, and 0.1% Triton X-100, along with 0.1% BSA, to create the enzyme activity system for drug screening.
Result
Suppression rate is calculated as follows.
Symbols:
- : Slope of Experimental Group (rate of reaction in the presence of the inhibitor).
- : Slope of Negative Control (rate of reaction without any inhibitor).
- : Slope of Positive Control (rate of reaction with a known inhibitor).
The suppression rate curve is plotted alongside the negative control curve, where the red curve represents the control curve, and the black curve represents the curve after suppression.
| Name | Suppression Rate | Resource |
|---|---|---|
| jqez5 |  Suppression Rate: 43% | A synthetic EZH2 inhibitor that can be used for targeted therapy in lung cancer and other tumors. |
| PROTAC ERRα ligand 2 |  Suppression Rate: 49% | PROTAC ERRα Degrader-2 is a compound composed of an MDM2 ligand, a linker, and an ERRα binding moiety. |
| thz2 |  Suppression Rate: 44% | A synthetic compound used for the study of cyclin-dependent kinase (CDK) inhibition. |
| fiauridine |  Suppression Rate: 61% | Microbial metabolites, plant, or animal extracts. |
| t-5224 |  Suppression Rate: 47% | Inhibiting the invasion and metastasis of liver cancer cells through chemical synthesis. |
| vtp-27999 tfa |  Suppression Rate: 44% | It is a synthetic selective renin inhibitor used for the treatment of hypertension and related diseases. |
| LQZ-7i |  Suppression Rate: 71% | A chemical synthetic inhibitor targeting survivin, which can effectively suppress the growth of tumor cells. |
| GFB8438 |  Suppression Rate: 66% | It is an effective TRPC5 inhibitor with good selectivity and in vitro activity, synthesized chemically. |
| BMV-8c3o |  Suppression Rate: 41% | A chemically synthesized small molecule used for studying biological activity or pharmacological effects. |
In vitro Fluorescent Reporting System
To test the cleavage of target sequences by proteases in the fluorescent reporter system in vitro, and to observe the reconstitution of the two protein segments in the Flip system after cleavage, we purified FlipGFP beta sheet 1-9 and beta sheet 10-11 separately. Each specific protease target cleavage sequence requires a different beta sheet 10-11, as the target sequence is inserted within this region.
When testing the FlipGFP fluorescent reporter system in vitro, we conducted the experiment in three stages, each involving a different target cleavage sequence. These included one TEV protease cleavage sequence and two V8 protease cleavage sequences, which provided guidance for the experimental design of subsequent in vivo validation.
TEV Protease Cleavage
First, we utilized the target cleavage sequence of TEV protease ENLYFQS, which consists of seven amino acids recognized specifically by the TEV protease. Given that the activation of the FlipGFP system by TEV protease in HEK293 cells has been documented in the literature, and TEV protease cleavage is known for its high specificity, employing TEV protease initially allows us to optimize experimental conditions such as protein purification, cleavage time, and assembly time. This lays a solid foundation for subsequent experiments.
For the protein purification results, we first performed SDS-PAGE analysis on the supernatant, precipitate, and samples before and after washing, as well as the elution fractions from the purification process. Based on protein size and the electrophoresis results, we identified the target proteins FlipGFP beta 1-9 and FlipGFP beta 10-11 (with a TEV protease cleavage site).
Since the molecular weight displayed in the SDS-PAGE results may differ slightly from the calculated value, we further conducted mass spectrometry to confirm that the proteins in the bands matched the designed sequences. It is important to note that a sequence coverage exceeding 90% in mass spectrometry can be considered as confirming that the protein sequence matches the design. This is a very high coverage, as mass spectrometry rarely achieves 100% coverage.
The purified beta10-11 fragment was incubated with TEV protease at 4°C for 1 hour, with an enzyme-to-substrate ratio of 1:100. After incubation, a 200 μL fluorescent protein self-assembly system was prepared, with FlipGFP beta1-9 at a concentration of 2.9 μM and FlipGFP beta10-11 at 6.5 μM. Following the initiation of the assembly process, absorbance at 535 nm was measured every hour using a plate reader, with excitation at a wavelength of 488 nm. Absorbance was recorded over a total period of 12 hours.
The results from the microplate reader show that, over the course of the assembly process, the fluorescence intensity in the experimental group (which includes successfully cleaved Flip GFP beta sheet 10-11 with the TEV protease cleavage site and Flip GFP beta 1-9) steadily increased. In contrast, the fluorescence intensity in the control group (which contains the same concentration of Flip GFP beta sheet 10-11 with the TEV protease cleavage site and Flip GFP beta 1-9 but without cleavage) showed almost no change over time.
V8 Protease Cleavage
Following the successful validation of using the TEV protease for specific cleavage of target sequences, in our next experiment, we will replace the target cleavage sequence with that of V8 protease. For the V8 protease cleavage sequence, we selected the extracellular segment of the PAR1 receptor, which is known to be cleaved by V8 protease and involved in triggering itch sensations in humans. This is in line with our goal of designing an itch-sensing system for in vivo applications.
For the protein purification results, we first performed SDS-PAGE analysis on the supernatant, precipitate, and samples before and after washing, as well as the elution fractions from the purification process. Based on protein size and the electrophoresis results, we identified the target proteins FlipGFP beta 10-11 (with a V8 protease cleavage site).
Similar to the target cleavage sequence using TEV protease, we also performed mass spectrometry analysis on this part. The results showed that we successfully purified the correct FlipGFP beta sheet 10-11 (V8 cleavage site) protein.
The results from the microplate reader show that, over the course of the assembly process, the fluorescence intensity in the experimental group (which includes successfully cleaved Flip GFP beta sheet 10-11 with the V8 protease cleavage site and Flip GFP beta 1-9) steadily increased. In contrast, the fluorescence intensity in the control group (which contains the same concentration of Flip GFP beta sheet 10-11 with the V8 protease cleavage site and Flip GFP beta 1-9 but without cleavage) showed almost no change over time.
V8-short cleavage site
We are optimizing the inserted V8 protease target cleavage sequence in the Flip system. Given that V8 protease target sequences do not exhibit strong specificity (its cleavage sites include aspartic acid and glutamic acid), we have selected a shorter sequence that contains only one cleavage site in the middle, reducing its length to one-third of the original sequence. This adjustment aims to minimize potential non-specific cleavage during future expression in Escherichia coli. Additionally, the shorter sequence helps prevent the elimination of spatial hindrance between the beta sheets 10-11 of uncut FlipGFP due to an overly long flexible sequence. Such a scenario could lead to unintended self-assembly and fluorescence in the uncut state.
For the protein purification results, we first performed SDS-PAGE analysis on the supernatant, precipitate, and samples before and after washing, as well as the elution fractions from the purification process. Based on protein size and the electrophoresis results, we identified the target proteins FlipGFP beta 10-11 (with a V8-short protease cleavage site).
Similar to the target cleavage sequence using TEV protease, we also performed mass spectrometry analysis on this part. The results showed that we successfully purified the correct FlipGFP beta sheet 10-11 (V8-short cleavage site) protein.
After shortening the V8 protease target cleavage sequence, we observed similar fluorescence intensity compared to the original sequence (with all other experimental conditions remaining unchanged). Therefore, we believe the shortened V8-short cleavage site can be used as the final target cleavage sequence for in vivo experiments with V8 protease.
In vivo Pruritus Fluorescent System
TEV Protease Cleavage
To create an in vivo Flip fluorescent reporter system, we started by testing FlipCherry. Given that the fluorescent protein was modified, we initially validated it using TEV protease. This involved inserting the TEV protease target cleavage sequence into the FlipCherry beta10-11 segment, allowing us to confirm the modified system’s functionality before proceeding with further tests.
To streamline experimental procedures, we aimed to express the entire system from a single plasmid. We selected the high-copy pRSFDuet-1 plasmid from the Duet series as our template, which contains two T7 promoters for simultaneous expression of two gene segments. Using Gibson Assembly, we inserted FlipCherry beta1-9, FlipCherry beta10-11 (with the TEV cleavage site), and EGFP (as a fluorescent control) after the first T7 promoter. These gene segments were linked using T2A sequences, which enable the production of three independent proteins from a single, continuous sequence by inducing a ribosomal “skip” during translation. This results in separate translation of FlipCherry beta1-9, FlipCherry beta10-11 (with the TEV cleavage site), and EGFP, despite being linked in the sequence. For the second T7 promoter, we used the same Gibson Assembly method to insert the TEV protease gene, optimizing its codons for E. coli expression.
After constructing the plasmid, we introduced it into BL21 (DE3) for expression and culture. When the bacterial culture reached an OD600 range of 0.6 to 1.0 after passage, we initiated protein expression using various concentrations: 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM. Following 12 hours of induction, we used a fluorescence microscope and a microplate reader to measure fluorescence results. The data obtained from these instruments confirmed the viability of the Flipcherry system for expressing protease cleavage activity in E. coli. Building on this successful validation, we developed an E. coli platform to investigate the cleavage activity of V8 protease.
- Experiment Group
- Control Group
V8-short Cleavage Site
Due to the short length of the substituted V8 protease cleavage site and its homologous protease cleavage site, it is difficult to obtain the correct fragment using traditional Gibson Assembly methods. Therefore, we directly attached the sequence to be replaced onto the primer of the vector, incorporating it as part of the vector’s homologous arm in the plasmid. We then used standard Gibson Assembly to replace the TEV protease with Glutamyl endopeptidase (gseA), successfully constructing the desired plasmid.
We had GeneScript directly synthesize the thermolysin gene sequence on the pETDuet-1 vector, completing the construction of one of the co-transformation plasmids. Both plasmids are regulated by a lactose operon system.
After constructing the plasmid, we introduced it into BL21 (DE3) for expression and culture. Once the bacterial culture reached an OD600 between 0.6 and 1.0, protein expression was induced using varying concentrations: 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM. Following 12 hours of induction, fluorescence was measured with a fluorescence microscope under consistent conditions. The microscope images captured under identical settings revealed similar green fluorescence in both the control and experimental groups. The control group lacked the gseA sequence following the second T7 promoter. EGFP served as an internal reference and was expressed in the plasmids of both groups. However, differences were observed in red fluorescence; the experimental group displayed red fluorescence due to self-assembly after cleavage, a phenomenon not seen in the control group.
- Experiment Group
- Control Group
