Objectives
The primary objective of this proof of concept is to establish a robust experimental framework aimed at identifying substances that can effectively alleviate pruritus caused by Staphylococcus aureus. This framework leverages the V8 protease mechanism, which is implicated in the itching sensation in humans.
Methodology
The methodology is structured around the DBTL (Design, Build, Test, Learn) cycle, ensuring a systematic, iterative approach to refining our experimental setup. We outline four main components of the PoC: V8 Protease Purification, Drug Screening, Flip Reporting System Verification and Simulation of Pruritus Mechanism in E. coli.
V8 Protease Purification
Design
Based on previous studies, V8 protease is a challenging enzyme to purify due to its propensity for self-degradation in recombinant E. coli systems. To overcome this, four specific amino acid mutations were introduced into the prosequence of the V8 protease to inhibit self-degradation. The mutated proenzyme was designed to carry a C-terminal His-tag for purification purposes.
Build
The gene encoding the mutant V8 protease was cloned into the pET-28a vector and transformed into E. coli BL21(DE3) cells.
Test
The protein was successfully expressed and purified. Initial assays showed a shift in protein bands upon cleavage, confirmed by mass spectrometry. However, protease activity was not detected, likely due to residual non-covalent attachment of the prosequence.
Learn
A more efficient purification method involving anion exchange chromatography (Q column) was implemented, which successfully separated the prosequence, yielding active V8 protease as confirmed by fluorescence assays.
Drug Screening
Design
A high-throughput screening (HTS) system was set up using a library of 3,600 small molecule compounds. The goal was to identify inhibitors of V8 protease activity.
Build
Using HTS technology, the compounds were screened against the active V8 protease.
Test
Nine compounds were identified as potential inhibitors, with inhibition rates ranging from 40% to 71%.
Learn
While initial results were promising, the inhibition rates were not sufficiently high. Expanding the screening library and iterating on the conditions will be necessary.
Flip Reporting System Verification
Design
A flip fluorescent protein system based on previous research was designed to report the cleavage activity of V8 protease. Initially, the system was tested with TEV protease to validate the cleavage mechanism.
Build
A 6x His-tagged FlipGFP system (beta1-9 and beta10-11 with the TEV target sequence) was constructed and expressed in E. coli.
Test
Initial in vitro tests did not show significant fluorescence. Adjustments were made to cleavage times and protein concentrations, leading to successful validation of the system with TEV protease.
Learn
Concentration ratios and cleavage times are critical to the system’s performance. A shorter V8-specific cleavage sequence was designed to prevent non-specific assembly and minimize spatial hindrance.
Simulation of Pruritus
Design
The goal was to simulate itching by S. aureus in E. coli using a flip fluorescent system with the V8 protease target sequence substituted for the TEV sequence. A system to express V8 protease homolog, Glutamyl endopeptidase (gseA), was designed due to its similar cleavage specificity and better folding characteristics.
Build
Plasmids were constructed to co-express the fluorescent reporter system (FlipCherry, modified for target sequence of V8 protease) and the gseA protease in E. coli.
Test
Tests showed successful co-expression and specific red fluorescence indicative of cleavage activity, validated by fluorescence microscopy and plate reader measurements.
Learn
The system successfully simulates pruritus in E. coli, with fluorescence results indicating cleavage activity of gseA. Further optimization will focus on refining expression conditions to ensure robust and reproducible results.
Conclusion & Perspectives
Conclusion
The proof of concept successfully establishes a methodological framework for identifying and characterizing substances that can alleviate pruritus by targeting the V8 protease mechanism in Staphylococcus aureus. Ongoing refinements and iterative testing will enhance the system’s robustness, paving the way for potential therapeutic development.
Future Directions
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Expand Drug Screening: Increase the library of compounds and optimize screening conditions to identify more potent inhibitors.
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Optimize Expression Systems: Refine the E. coli expression systems to enhance the robustness and stability of the reporter proteins.