Comparison of the efficacy of cell wall disruption methods for Saccharomyces cerevisiae INVSc1 cells expressing Toxoplasma gondii recombinant ROP6 protein.

Toxoplasma gondii infects both humans and animals, causing severe clinical manifestations. Due to the lack of a safe vaccine, the development of a safe and effective vaccine against toxoplasmosis remains a significant need. Saccharomyces cerevisiae is a well-established recombinant protein expression system with Generally Recognized as Safe status. It supports post-translational glycosylation and is employed in several licensed vaccines, including hepatitis B (Engerix-B), human papillomavirus (Gardasil), and malaria (Mosquirix). Recombinant T. gondii proteins expressed in S. cerevisiae have been explored for use in vaccine formulations. However, cell disruption during downstream processing presents a major challenge due to the robust cell wall of yeast. In our previous work, T. gondii recombinant ROP6 protein was successfully expressed in S. cerevisiae INVSc1 cells for use as a vaccine antigen, and cell disruption was performed using a microfluidizer. In this study, we compared the efficiency of four disruption methods (microfluidizer, acid-washed glass beads, liquid nitrogen, and Y-PER reagent) for releasing recombinant protein. The results demonstrated that the microfluidizer and acid-washed glass bead methods achieved superior cell wall disruption efficiency, making them suitable for large-scale processing. The liquid nitrogen method caused excessive protein degradation and presents safety and scalability challenges. Although Y-PER reagent yielded lower amounts of rROP6 protein compared to microfluidizer and acid-washed glass bead methods, it provided good protein stability by reducing multimerization and degradation and offered operational simplicity. Overall, the choice of disruption method should be guided by the target protein's characteristics, production scale, and cost considerations to optimize yield and stability.