Development of USP7 Full-Length Protein in E. Coli for Enhanced HTRF Assays

Ubiquitin-specific protease 7 (USP7), an important enzyme in the ubiquitin-proteasome system. This system is responsible for degrading unneeded or damaged proteins by tagging them with ubiquitin, a small protein, and then breaking them down in the proteasome. USP7 specifically plays a role in removing these ubiquitin tags, which can save proteins from degradation. USP7 is known for its role in regulating the p53 protein, a key tumor suppressor. It does so by deubiquitinating p53, thus preventing its degradation. Since p53 is crucial for preventing cancer development by regulating cell cycle and apoptosis, the interaction between USP7 and p53 is highly significant. USP7 also interacts with other proteins involved in cancer, such as PTEN, another tumor suppressor, and Mdm2, a regulator of p53. By modulating these proteins, USP7 indirectly influences cancer progression. Moreover, USP7 plays a role in cell cycle regulation. Alterations in the cell cycle are a hallmark of cancer cells, and the involvement of USP7 in this process makes it an important focus in cancer research.  Given its crucial role in regulating proteins associated with cancer, USP7 is a promising target for cancer therapy. Inhibitors of USP7 could potentially restore the normal function of tumor suppressors like p53, leading to the suppression of tumor growth. The development of selective USP7 inhibitors is a key area of research. These inhibitors aim to specifically target the cancer cells while sparing normal cells, reducing potential side effects.

In a new study published in the Journal of Pharmaceutical and Biomedical Analysis by Associate Professor Yihui Song, Shu Wang , Min Zhao, and Professor Bin Yu from Zhengzhou University developed a full-length USP7 protein expressed in the E. coli prokaryotic system, for improved Homogeneous Time-Resolved Fluorescence (HTRF) assay, paving the way for more accurate and efficient high-throughput screening in drug discovery.

The team initiated their research by constructing a comprehensive model of full-length USP7. Utilizing advanced computational tools like homology modeling and AlphaFold predictions, they synthesized a detailed structural representation of USP7. This step was crucial as it revealed seven targetable pockets within the protein, essential for the identification and development of specific inhibitors. The researchers chose the E. coli prokaryotic system for expressing the full-length USP7 protein. This decision was strategic, given the system’s cost-effectiveness and efficiency compared to conventional eukaryotic expression systems. The authors cloned the USP7 gene into an appropriate vector and subsequently introduced into E. coli cells. The team meticulously optimized the expression conditions, including temperature, induction time, and culture medium, to maximize the yield of correctly folded and functional USP7 protein. Following expression, the USP7 protein underwent a rigorous purification process. This involved multiple steps, including affinity chromatography and size-exclusion chromatography, to ensure the extraction of a highly pure and active form of the protein. The purification process was critical to maintain the structural integrity and functional activity of USP7, which is essential for the assay’s accuracy.

One important development by the research team was the design of a HTRF assay. The assay was designed to measure the deubiquitinating activity of USP7 against a ubiquitin precursor, UBA10, thereby enabling the screening of potential inhibitors. The researchers undertook a systematic optimization of the assay. This included fine-tuning the concentrations of USP7, UBA10, and other assay components, along with optimizing buffer conditions and reaction times. Such meticulous calibration was essential to ensure the assay’s sensitivity and specificity. To validate the assay, the team used known USP7 inhibitors, including GNE-6776. The assay’s performance was benchmarked against previously reported data for these inhibitors, demonstrating its accuracy and consistency. The successful validation with known inhibitors was a critical step in proving the assay’s effectiveness in identifying potent USP7 inhibitors.

The researchers conducted an extensive analysis of the data obtained from the HTRF assay. This involved calculating the potency of various inhibitors, determining their IC₅₀ values, and analyzing their specificity towards USP7. Their results were compared with existing data on USP7 inhibitors, providing a comprehensive understanding of the assay’s efficacy in identifying and characterizing potential therapeutic agents. Through these meticulous and innovative methodologies, the researchers successfully developed a robust and reliable HTRF assay using the full-length USP7 protein expressed in E. coli. This breakthrough not only enhances the drug discovery process for USP7 inhibitors but also sets a precedent for the development of similar assays for other therapeutic targets. The study’s meticulous approach, from protein expression to assay development and validation, underscores the intricacy and precision required in advanced medicinal research.

The study’s implications are far-reaching and multifaceted. Firstly, the successful expression of USP7 full-length in E. coli is a game-changer in the field of drug discovery. This approach offers a more cost-effective and scalable alternative to traditional eukaryotic expression systems, making it a valuable tool for large-scale drug screening initiatives. Furthermore, the novel HTRF assay developed in this study addresses several limitations of previous methodologies. By incorporating the full-length USP7 protein, the assay enhances the specificity and selectivity of inhibitor screening, a critical factor in developing effective therapeutic agents. This advancement is particularly significant in the context of cancer therapeutics, where precision and specificity are paramount. Additionally, the introduction of this novel HTRF assay model could have broad implications across various therapeutic areas. Its adaptability and efficiency make it a promising tool for the identification and development of inhibitors targeting a wide range of proteins implicated in various diseases, thereby expanding the scope and impact of drug discovery research.