Overview

In order to successfully realize our product design, which aims to identify substances that can effectively alleviate pruritus based on the mechanism by which Staphylococcus aureus induces itching in humans, we have established three primary objectives in our experimental design:

1. Identify substances with the potential to inhibit the pruritus-inducing activity of V8 protease.
2. Verify the feasibility of the flip fluorescent reporting system in vitro .
3. Express the complete pruritus simulation system in E. coli.

The entire engineering process is logically structured under the principles of DBTL (Design, Build, Test, Learn), as recommended by iGEM. We continuously conduct transformations and iterations to refine our projects. Below is a summary of the experimental content and the logical workflow we have completed.

V8 Purification

Research

Based on previous studies, V8 protease, a zymogen, consists of three parts: the preprosequence, the prosequence, and the mature sequence. The preprosequence encodes a signal peptide, while the prosequence deactivates the enzyme activity of V8 protease and assists in proper folding. The catalytic site of the enzyme is located within the mature sequence.

V8 protease is a zymogen.

When wild-type V8 protease is directly expressed in E. coli BL21(DE3), it undergoes self-degradation, resulting in very low protein yield and making the enzyme difficult to obtain. However, introducing four mutations into the prosequence of the V8 protease or replacing it with the prosequence of the homologous protein GluSE can inhibit self-degradation. In this study, we opted to introduce four specific amino acid mutations into the prosequence to obtain the protein. The natural form of V8 protease requires cleavage of the prosequence for activation, and thermolysin has been demonstrated to effectively activate it.¹

Cycle 1

• Design

  To express the V8 proenzyme and activate it via in vitro cleavage with thermolysin to produce active V8 protease for drug screening, we constructed the sspA-Mut4-his(C) plasmid within an E. coli expression system, aiming to obtain the active protein.

  

  Inhibition of Self-Degradation of V8 Protease

• Build

  The gene encoding the V8 protease, lacking the preprosequence and containing four amino acid mutations in the prosequence, was engineered to include a His-tag at the C-terminus. This design aims to prevent self-degradation of the protein in E. coli.

  

  pET-28a-His(C)-pro-sspA-mut4 Map

• Test

  The V8 proenzyme was successfully obtained. Upon cleavage, the protein band shifted from the sixth to the fifth position on the gel. Mass spectrometry confirmed that the molecular mass matched the expected value (noting that mass spectrometry disrupts non-covalent interactions), and peptide fingerprinting revealed detectable fragments of the prosequence. However, no protease activity was detected in this round of testing.

• Learn

  The prosequence may have remained non-covalently attached to the mature protein post-cleavage, potentially interacting with the active site and inhibiting V8 protease activity. Implementing a more effective purification method might be necessary to isolate active V8 protease.

Cycle 2

• Design

  We hypothesized that anion exchange chromatography (Q column) would be more effective in purifying the V8 protease by separating the non-covalently attached prosequence from the mature enzyme, thereby yielding active V8 protease.

Q column

• Build

  After activating the V8 protease through enzymatic cleavage, we used an anion exchange column to attempt the separation of the prosequence from the V8 protease.

  

  Activation of V8 protease by thermolysin

• Test

  Following passage through the Q column, the protein band shifted from the fifth to the fourth position on the gel. Fluorescence assays using a V8 protease-specific substrate demonstrated a linear increase in fluorescence over time during incubation, confirming the acquisition of active V8 protease.

Drug Screening

• Design

  Using high-throughput drug screening, we tested 3,600 small molecule compounds from the Natural Compound Library, Bioactive Compound Library, Clinical Compound Library, and Approved Drug Library for their inhibitory effects on V8 protease activity.

• Build

  Using high-throughput screening technology, we screened a diverse library of small molecule compounds to identify potent inhibitors of V8 protease activity.

Drug Screening in the Lab

• Test

  Among 3,600 small molecule compounds, we identified nine with a high inhibition rate (range from 40% to 71%) against V8 protease activity, making them potential lead compounds for its inhibitors.

• Learn

  The nine potential inhibitors identified from over 3,600 compounds showed limited inhibition rates. As a next step, we plan to expand the screening scope to identify small molecules with higher inhibition rates.

Flip Reporting System in vitro Verification

Research

Based on previous research, a lab at the University of California, San Francisco, developed a protease-activated fluorescent reporter system. In this system, the structure of the fluorescent protein is divided into two parts: beta sheets 1-9 and beta sheets 10-11. Initially, one of the beta strands in the latter part is flipped. The two beta strands of beta sheets 10-11 are connected by a linker, which contains the target cleavage sequence for the protease. Upon activation of the protease, the linker in beta sheets 10-11 is cleaved, allowing the flipped beta strand to return to its correct orientation and assemble with beta sheets 1-9, forming a complete fluorescent protein structure that emits light. This mechanism of the fluorescent reporter system inspires us to use it to characterize the cleavage activity of V8 protease on its target sequence, thereby simulating the human itching sensation in vivo.²

The principle of the "Flip" protease fluorescent reporter system

Cycle 1

• Design

  To validate the feasibility of the Flip system, we first conducted in vitro tests using the well-established TEV protease. After purifying the proteins, we confirmed the sequence accuracy through mass spectrometry. Next, we incubated TEV protease with beta10-11 to ensure complete cleavage of the target sequence. This step minimizes any potential variability in the cleavage process that could affect the subsequent self-assembly of beta1-9 with the cleaved beta10-11 into the superfold GFP fluorescent protein.³

• Build

  We added a 6x His-tag to the C-terminus of both parts of the fluorescent system (beta1-9 and beta10-11) for expression and purification. We selected the same FlipGFP sequence and TEV protease target cleavage sequence as described in the literature. The TEV protease target sequence is ENLYFQS. These sequences were submitted to GeneScript for direct synthesis in the expression region of the pET-28a plasmid.

  

  Plasmids for the FlipGFP system
  

  Plasmids for the FlipGFP system

• Test

  The two plasmids were separately transformed into BL21 (DE3) cells for expression. When the OD600 of the culture reached 0.6, IPTG was added at a final concentration of 0.1 mM to induce protein expression, and the cultures were incubated for 14 hours before harvesting the cells for subsequent purification steps. 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.

• Learn

  During the 12-hour assembly process, no significant fluorescence change was observed. Since mass spectrometry confirmed the accuracy of the purified protein sequences, we hypothesize that the issue may be due to the high cleavage efficiency of the TEV protease and the relatively long incubation time, which could have led to incorrect cleavage, preventing the fluorescent protein from successfully self-assembling into a functional structure. Therefore, a higher concentration of correctly cleaved beta10-11 fragments is needed.

Cycle 2

• Design

  After protein electrophoresis and mass spectrometry analysis, we confirmed that the purified protein had no sequence errors. Therefore, we aimed to improve the self-assembly process by adjusting the experimental conditions. We shortened the TEV protease cleavage time to avoid potential nonspecific cleavage and increased the concentration of the FlipGFP beta sheet 10-11 fragment during assembly to ensure sufficient fragments were available for assembly with FlipGFP beta sheet 1-9.

• Test

  The two plasmids were separately transformed into BL21 (DE3) cells for expression. When the OD600 of the culture reached 0.6, IPTG was added at a final concentration of 0.1 mM to induce expression, and the cultures were incubated for 14 hours before collecting the cells for subsequent purification steps. The purified beta10-11 fragment was incubated with TEV protease at 4°C for 5 minutes, using enzyme-to-substrate ratios of 1:50 and 1:100. After incubation, a 200 μL fluorescent protein self-assembly system was prepared, with the concentration of FlipGFP beta1-9 at 5.8 μM and FlipGFP beta10-11 at 44.6 μM. Following the initiation of the assembly process, absorbance at 535 nm was measured using a plate reader every hour for 11 hours, with excitation at a wavelength of 488 nm.

Result of FlipGFP(TEVprotease) in vitro test

• Learn

  We successfully validated the FlipGFP fluorescent reporter system. Based on the in vitro results, we realized that the concentration ratio between the two protein segments in the Flip system has a significant impact on the experimental outcome. Therefore, in future in vivo experiments, it will be essential to carefully control the induction time and protein concentrations to ensure optimal protein assembly.

Cycle 3

• Design

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.

• Build

  We construct the plasmid with V8 protease cleavage site inserted in FlipGFP beta sheet 1-9.

  

  Plasmid for FlipGFP(V8)

• Test

  The two plasmids were separately transformed into BL21 (DE3) cells for expression. When the OD600 of the culture reached 0.6, IPTG was added at a final concentration of 0.1 mM to induce expression, and the cultures were incubated for 14 hours before collecting the cells for subsequent purification steps. The purified FlipGFP beta sheet 10-11 fragment was incubated with V8 protease at 4°C for 5 minutes, using enzyme-to-substrate ratios of 1:50 and 1:100. After incubation, a 200 μL fluorescent protein self-assembly system was prepared, with the concentration of FlipGFP beta sheet 1-9 at 5.8 μM and FlipGFP beta sheet 10-11 at 44.6 μM. Following the initiation of the assembly process, absorbance at 535 nm was measured using a plate reader every hour for 3 hours, with excitation at a wavelength of 488 nm.

Result of FlipGFP(V8protease) in vitro test

• Learn

We have successfully validated the feasibility of using the Flip system to report V8 protease activity. Building on this foundation, we are now optimizing the Flip system to ensure its robust expression in Escherichia coli.

Cycle 4

• Design

  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.

• Build

  We construct the plasmid with V8 protease short cleavage site inserted in FlipGFP beta sheet 1-9. This is based on the former plasmid, pET-28a-FlipGFP(ß10-11)-V8protease. We did Gibson Assembly to substitute V8 protease cleavage site by adding the V8-short cleavage site directly on the primers of the vector.

  

  Plasmid for FlipGFP(V8)

• Test

  The two plasmids were separately transformed into BL21 (DE3) cells for expression. When the OD600 of the culture reached 0.6, IPTG was added at a final concentration of 0.1 mM to induce expression, and the cultures were incubated for 14 hours before collecting the cells for subsequent purification steps. The purified FlipGFP beta sheet 10-11 fragment was incubated with V8 protease at 4°C for 5 minutes, using enzyme-to-substrate ratios of 1:50 and 1:100. After incubation, a 200 μL fluorescent protein self-assembly system was prepared, with the concentration of FlipGFP beta sheet 1-9 at 5.8 μM and FlipGFP beta sheet 10-11 at 44.6 μM. Following the initiation of the assembly process, absorbance at 535 nm was measured using a plate reader every hour for 3 hours, with excitation at a wavelength of 488 nm.

Result of FlipGFP(V8-short) in vitro test

• Learn

  Based on the experimental results, the use of a shorter V8 protease target cleavage sequence yields fluorescence outcomes comparable to those obtained with a longer target sequence. Taking into consideration the previously mentioned advantages—reduced non-specific cleavage and minimized spatial hindrance—we have decided to use the shorter V8 protease cleavage sequence as the insert for the FlipGFP system in subsequent experiments.

E.coli Pruritus Simulation System

Research

Through literature review, we identified the mechanism by which Staphylococcus aureus causes itching in humans: it secretes V8 protease, which cleaves the extracellular sequence of the PAR1 receptor on the skin. Based on this, we replaced the TEV protease cleavage sequence in the Flip system with the V8 protease target sequence to report the activity of V8 protease. To more accurately simulate the conditions of skin itching, we substituted FlipGFP with FlipCherry. FlipCherry contains a fluorescent protein structure based on the directed evolution of superfold Cherry, offering enhanced brightness.⁴

The mechanism by which human skin experiences itching upon exposure to Staphylococcus aureus.

V8 protease, also known as Glutamyl endopeptidase from Staphylococcus aureus, specifically cleaves peptide bonds at the carboxyl side of glutamic acid residues, making it a valuable tool in protein mapping. However, its tendency to misfold and self-degrade when expressed in non-native systems like E. coli presents challenges for experimental setups. By using Glutamyl endopeptidase as a substitute, we can preserve the same specificity at cleavage sites while avoiding the folding and degradation issues. This ensures that the cleavage analysis more accurately reflects the system’s behavior without introducing artifacts due to enzyme instability.

Detecting the efficacy of substances in a fluorescent reporter system simulating human itch sensation in Escherichia coli

Cycle 1

• Design

  In designing an in vivo Flip fluorescent reporter system, we first tested FlipCherry. Since the fluorescent protein was modified, we initially used TEV protease for validation by inserting the TEV protease target cleavage sequence into the FlipCherry beta10-11 segment. This allowed us to confirm the functionality of the modified system before moving forward with further testing.

• Build

  To simplify experimental procedures, we aimed to express the entire system from a single plasmid. Therefore, we selected the high-copy pRSFDuet-1 plasmid from the Duet series as the template. This plasmid contains two T7 promoters, allowing for the simultaneous expression of two gene segments. After the first T7 promoter, we inserted FlipCherry beta1-9, FlipCherry beta10-11 (with the TEV cleavage site), and EGFP (as a fluorescent control) using Gibson Assembly. The three gene segments were linked using T2A sequences. The T2A sequence enables the production of three independent proteins from a single, continuous sequence by inducing a ribosomal “skip” during translation. As a result, although the genes are linked in the sequence, they are translated into separate proteins: FlipCherry beta1-9, FlipCherry beta10-11 (with the TEV cleavage site), and EGFP. This approach allows for efficient co-expression of multiple proteins from a single mRNA transcript. Similarly, after the second T7 promoter, we inserted the TEV protease gene, optimizing its codons for E. coli expression using the same Gibson Assembly method.

Plasmid pRSFDuet-Flipcherry(TEV)-EGFP-TEVprotease for in vivo Flipcherry system Testing

• Test

  After constructing the plasmid, we transferred it into BL21 (DE3) for expression and passage. After the passage, when the bacterial culture reached an OD600 range of 0.6–1.0, we induced protein expression at varying concentrations: 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM. After 12 hours of induction, the fluorescence results were measured using a fluorescence microscope and a microplate reader.

• Learn

  Based on the data obtained from the fluorescence microscope and microplate reader, we successfully validated the feasibility of the Flipcherry system in expressing protease cleavage activity in E. coli. Building on this system, we further developed an E. coli platform to explore the cleavage activity of V8 protease.

Cycle 2

• Design

  Due to the multiple cleavage sites of V8 protease in the extracellular sequence of PAR1 and the fact that this extracellular sequence is as long as 81 amino acids, it may influence the structure of other parts of the beta10-11 region. Therefore, we extracted only a segment of 17 amino acids from the extracellular sequence of PAR1 as the target cleavage sequence for V8 protease, replacing the original V8 protease cleavage sequence in the plasmid.

  At the same time, when mature V8 protease is expressed alone in E. coli, it may not fold correctly and can undergo self-degradation. This phenomenon can hinder accurate assessment of the protease’s activity. Therefore, to better validate the system’s representation of V8 protease cleavage, we replaced V8 protease with its homologous protease, Glutamyl endopeptidase, in our experiments. Glutamyl endopeptidase shares the same cleavage sites as V8 protease and offers a comparable mechanism of action.

  

  V8 protease from Staphylococcus aureus cleaves the PAR1 receptor on the skin surface.

  Protein gseA includes signal peptide and propeptide. Through research¹, we discovered that adding a signal peptide can help gseA fold correctly and reduce its toxicity in vivo. Once gseA has folded properly, thermolysin can be used to remove the signal peptide, thereby activating the gseA. Based on this, we designed a co-transformation strategy in E. coli, introducing plasmids carrying both the Flipcherry reporter system and gseA with a signal peptide, along with a plasmid expressing thermolysin. We selected pETDuet-1 and pRSFDuet-1 plasmids, as this pair has been validated to successfully co-express in E. coli.

The 3D structure of gseA(Glutamyl endopeptidase)

• Build

  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.

  

  Plasmid pRSFDuet-Flipcherry(V8short)-EGFP-gseA for in vivo Flipcherry system Testing

  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.

  

  Plasmid pETDuet-thermolysin for in vivo Flipcherry system Testing

• Test

  After constructing the plasmid, we transferred it into BL21 (DE3) for expression and passage. After the passage, when the bacterial culture reached an OD600 range of 0.6–1.0, we induced protein expression at varying concentrations: 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM. After 12 hours of induction, the fluorescence results were measured using a fluorescence microscope (under the same conditions).

  

  Fluorescence Result of Experimental Group
  

  Fluorescence Result of Control Group

• Learn

  Fluorescence microscope images were obtained under identical imaging parameters, showing comparable green fluorescence in both the control group (where no gseA sequence was added after the second T7 promoter) and the experimental group. EGFP, used as an internal reference, was expressed in the plasmids of both groups. However, the red fluorescence differed between the two. In the experimental group, red fluorescence induced by self-assembly after cleavage was observed, while this phenomenon was absent in the control group.

  These experimental results suggest the feasibility of using the Flipcherry fluorescence reporting system to characterize the cleavage activity of gseA. This system successfully simulates the process in E. coli that mimics the itching sensation on human skin caused by Staphylococcus aureus.