Abstract：
Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2\'s biochemical properties, providing valuable insights into its serine protease domain.
摘要：
跨膜丝氨酸蛋白酶2(TMPRSS2)是属于II型跨膜丝氨酸蛋白酶(TTSP)家族的膜结合蛋白酶。它是一种多域蛋白质，包括负责其自我激活的丝氨酸蛋白酶结构域。该蛋白质被认为是致癌转录因子，并且具有切割（引发）SARS-CoV-2刺突蛋白的能力。为了表征TMPRSS2的生化特性，我们使用pPICZαA载体在Komagataellaphafii中表达了丝氨酸蛋白酶结构域（rTMPRSS2_SP），并使用固定金属亲和（NiSepharose™excel）和大小排阻（Superdex75）色谱对其进行了纯化。我们探索了可操作的荧光共振能量转移FRET肽作为底物。我们选择了Abz-QARK-（Dnp）-NH2（Abz=邻氨基苯甲酸，荧光供体，Dnp=2,4-二硝基苯，淬灭剂基团）作为底物，以找到最大酶活性的最佳条件。我们发现，金属离子，如Ca2+和Na+增加酶活性，但离子表面活性剂和还原剂降低了催化能力。最后,我们确定了长期储存的rTMPRSS2_SP稳定性。总之,我们的结果代表了TMPRSS2的生化特性的第一个全面表征，提供对其丝氨酸蛋白酶结构域的有价值的见解。
