Comprehensive Investigation of Recombinant Human TMPRSS4 Expression and Purification Across Diverse Expression Platforms: Bacterial, Insect (BVES), and Mammalian Systems

Abstract

TMPRSS4, an essential transmembrane protease serine 4, holds significant relevance in diverse biological contexts, making it a molecule of interest across various fields. This transmembrane serine protease, TMPRSS4, plays a pivotal role in multiple areas, including cancer research, disease processes, and potentially in the spread of viral infections.  In light of its multifaceted significance, the research and production of active TMPRSS4 protein serve as a critical foundation for in-depth structural and functional studies, ultimately leading to the identification of potential inhibitors. Within this context, our study encompasses an extensive investigation of various expression systems, including microbial, insect cells, and mammalian cells, aimed at obtaining biologically active TMPRSS4 protein. This study underscores our efforts in optimizing solubility. We explored different E. coli expression strains, optimized expression conditions at various temperatures, employed both inducible and autoinduction-based approaches, fine-tuned the Multiplicity of Infection (MOI), and controlled cell density in Sf9 cells using BVES. Additionally, we delved into the exploration of alternative signal peptides for secretory expression in mammalian cells, followed by the characterization of purified protein variants. Through rigorous biochemical analyses and functional assays, we discovered the distinct advantages associated with mammalian cell expression, ultimately resulting in the production of biologically active TMPRSS4. Our findings provide critical insights into the optimization of expression systems, thereby enhancing the functionality of the protein.