Overview
Our project addresses the challenge of producing active V8 protease through advanced genetic and purification strategies. V8 protease, a zymogen prone to self-degradation in E. coli, complicates production and yields. We designed a pET-28a expression plasmid, sspA-Mut4-his(C), introducing four mutations in the prosequence to prevent degradation and employed thermolysin for activation. Initial attempts showed no activity due to non-covalent attachment of the prosequence. By purifying the enzyme with anion exchange chromatography, we produced active V8 protease, confirmed by fluorescence assays.
Moreover, we adapted a protease-activated fluorescent reporter system from UC San Francisco, replacing the TEV protease sequence with that of V8 protease for in vitro validation. We optimized the cleavage conditions and tested with FlipCherry for better brightness. Despite early challenges, subsequent conditions led to successful validation. Issues with V8 protease stability were resolved by using Glutamyl endopeptidase (gseA) in a co-expression system.
Finally, for drug screening, we used high-throughput methods and computational tools, identifying 60 promising inhibitors from virtual screening. Although experimental validation was delayed, our multi-faceted approach sets a solid foundation for large-scale enzyme production, drug discovery, and further research on conditions mediated by V8 protease.
Protein Purification
In our project, we aimed to address the challenge of producing active V8 protease by utilizing advanced genetic and purification strategies. V8 protease, a zymogen, is composed of three distinct segments: the preprosequence, the prosequence, and the mature sequence. The preprosequence contains a signal peptide, while the prosequence functions in deactivating enzyme activity and aiding proper folding. The active catalytic site is located within the mature sequence. A major issue with expressing wild-type V8 protease in E. coli BL21(DE3) is self-degradation which leads to low protein yields. Previous studies have indicated that introducing specific mutations into the prosequence or replacing it with a sequence from a homologous protein like GluSE can mitigate this problem. Hence, we decided to introduce four specific mutations into the prosequence to prevent degradation and subsequently activate the zymogen using thermolysin.
In the first cycle of our experiment, we designed a pET-28a expression plasmid, named sspA-Mut4-his(C), to express the V8 proenzyme in E. coli. The gene encoding the V8 protease was modified to lack the preprosequence but include four mutations in the prosequence, along with a C-terminal His-tag, to protect against self-degradation. We successfully expressed the V8 proenzyme and verified its production through gel electrophoresis, which showed that the protein band shifted position after cleavage by thermolysin. Mass spectrometry confirmed the anticipated molecular mass of the cleaved product, suggesting proper cleavage had occurred. However, despite successful cleavage, no protease activity was observed, likely due to non-covalent attachment of the prosequence to the mature enzyme, which may have inhibited activity by blocking the active site.
In the second cycle, we addressed this issue by hypothesizing that anion exchange chromatography could effectively separate the mature enzyme from the residual prosequence. Post-activation with thermolysin, we purified the V8 protease using an anion exchange column (Q column). This process successfully shifted the protein band position, and subsequent fluorescence assays using a substrate specific to V8 protease confirmed active enzyme production, as indicated by a steady increase in fluorescence over time during incubation.
Following successful purification and activation of the V8 protease, we proceeded to screen for potential inhibitors using high-throughput drug screening technologies. These experiments establish a method for expressing and obtaining active V8 protease by introducing specific mutations to prevent self-degradation and employing effective purification techniques. This approach not only provides a foundation for large-scale enzyme production but also sets the stage for drug discovery efforts aimed at identifying inhibitors of the V8 protease.
Protease Reporting System
A lab at the University of California, San Francisco, initially developed a protease-activated fluorescent reporter system, where a fluorescent protein is divided into two parts, beta sheets 1-9 and beta sheets 10-11. In this system, one beta strand in the beta sheets 10-11 is initially flipped. The two beta strands of beta sheets 10-11 are connected by a linker containing a target cleavage sequence for a protease. Upon protease activation, the linker in beta sheets 10-11 is cleaved, allowing the flipped beta strand to return to its correct position and assemble with beta sheets 1-9, forming a complete fluorescent protein structure that emits light. This mechanism inspired us to use the system to characterize V8 protease activity on its target sequence to simulate human itching sensation in vivo.
Aligning with this concept, we aimed to replace the TEV protease cleavage sequence in the Flip system with the V8 protease target sequence to report V8 protease activity, simulating a skin itching condition. We decided to use FlipCherry instead of FlipGFP for better brightness, as FlipCherry is based on the directed evolution of superfold Cherry.
Our validation began with FlipCherry using TEV protease. We confirmed the sequence through mass spectrometry and optimized the cleavage conditions. After purifying the proteins, we conducted in vitro tests which demonstrated that no significant fluorescence change occurred during the 12-hour assembly process, likely due to improper cleavage conditions and incorrect concentration of fragments. Adjusting these conditions in subsequent tests allowed successful validation of the FlipCherry system.
To simulate the itching caused by Staphylococcus aureus, which secretes V8 protease cleaving the PAR1 receptor sequence, we substituted the TEV protease cleavage site with that of V8 protease in our Flip system. We also faced challenges with V8 protease’s misfolding and self-degradation in E. coli. To overcome these issues, we replaced V8 protease with Glutamyl endopeptidase (gseA), a homologous protease with similar cleavage specificity but greater stability when expressed in E. coli. This approach preserved the cleavage site specificity while mitigating folding and degradation issues.
We constructed a plasmid using the pRSFDuet-1 vector to express the entire system from a single plasmid, driven by two T7 promoters. The first promoter expressed FlipCherry beta1-9, FlipCherry beta10-11 (with the target cleavage sequence), and EGFP as an internal reference, linked by T2A sequences to ensure production of separate proteins. The second promoter drove the expression of the TEV protease or Glutamyl endopeptidase gene, optimized for E. coli expression, using Gibson Assembly.
Following successful expression and validation of the FlipCherry system, we induced protein expression in E. coli BL21(DE3) cells and confirmed the functionality of the modified system using fluorescence microscopy and a microplate reader. In further experiments, using a segment of the PAR1 receptor with the cleavage site of V8 protease, we observed red fluorescence induced by self-assembly after cleavage in the experimental group but not in the control group, illustrating the system’s ability to report cleavage activity.
Lastly, to address V8 protease misfolding, we added a signal peptide to aid its proper folding, with thermolysin used to remove the signal peptide post-folding. A co-transformation strategy using pETDuet-1 and pRSFDuet-1 plasmids facilitated the co-expression of the FlipCherry system and proteases in E. coli, demonstrating a reliable platform for further investigations and inhibitor screening.
Overall, these experiments demonstrated the feasibility and functionality of using the FlipCherry fluorescence reporter system to characterize V8 protease activity, simulating the itching sensation caused by Staphylococcus aureus in humans. This platform offers a reliable basis for future research and high-throughput screening for potential inhibitors.
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.
However, we are not content with the results obtained in the lab. To identify more potential active compounds, we leveraged modern computational science to conduct virtual drug screening. By using Schrodinger and GVSrun tools, we design the grid box and screen against the ChemDiv molecule library, which comprises over 1,000,000 compounds.
We saved the top 1000 results from the aforementioned screening and, considering factors such as protein-ligand bonding, conformational matching, electronegativity compatibility, and strain energy, we ultimately identified 60 of the most promising small molecules capable of inhibiting the V8 protease. Although the lengthy drug procurement cycle prevented us from experimentally validating the virtual screening outcomes before the deadline of this year’s iGEM competition, based on the quantitative results, these small molecules hold considerable promise for achieving stronger inhibitory effects than those obtained from laboratory screenings.