Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease domain

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.

Keywords:

• Komagataella phaffii
• pPICZαA
• protease
• SARS-CoV-2

Acknowledgment

We thank Professor Prof. Dr. Giuseppe Caliendo (Department of Pharmacy, School of Medicine, University of Naples, Italy) for help with the recombinant TMPRSS2 gene.

Disclosure statement

No potential conflicts of interest were reported by the authors.

Data availability statement

All data used in this study are included in this publication.

Additional information

Funding

The study had financial support provided by Fundação de Amparo à Pesquisa do Estado de São Paulo, (FAPESP—Projects: 2018/13588-0 and 2020/14426-3). The study had financial support provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. The study had financial support provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq—306023/2022-9.