Department of Biomedicine, University of Bergen, Norway.
Department of Chemistry, University of Bergen, Norway.
PLoS One. 2024 Jun 25;19(6):e0294827. doi: 10.1371/journal.pone.0294827. eCollection 2024.
Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.
中性粒细胞蛋白酶 3（PR3）是慢性阻塞性肺疾病和囊性纤维化等炎症性肺病的重要药物靶点。针对 PR3 的药物发现工作需要活性酶进行体外特性分析，如抑制剂筛选、酶测定和结构研究。使用杆状病毒表达系统表达重组 PR3（rPR3）克服了从人血中获取酶的需求，并且还可以研究突变对酶活性和配体结合的影响。在这里，我们报告了使用杆状病毒表达系统表达重组 PR3（rPR3）。所描述的纯化和激活过程得到了高纯度和高活性的 PR3。通过使用基于荧光的酶测定法，比较了存在市售抑制剂时 rPR3 和人 PR3 的活性。纯化的 rPR3 与人源天然酶具有相当的活性，因此是体外酶学研究的合适替代物。此外，我们建立了一种基于表面等离子体共振的测定法，以确定 PR3 配体的结合亲和力和动力学。这些方法为旨在治疗肺部炎症的早期药物发现提供了有价值的工具。
