Abstract:
This study focuses on developing an optimized method for protein extraction from Pichia pastoris for use in cell-free protein synthesis (CFPS) systems, particularly to improve yields for therapeutic protein production. The research addresses the gap in efficient lysis methods for P. pastoris, which is notoriously difficult to lyse due to its cell wall composition. The primary aim of the paper was to evaluate different lysis methods and their effects on protein concentration in cell extracts to develop a more efficient and high-yielding CFPS platform.
The methodology involved growing P. pastoris cultures to high cell densities and testing various lysis techniques, including chemical, enzymatic, sonication, and high-pressure cell disruption using Constant Systems Equipment. Protein yield was assessed by the DC protein assay, and CFPS performance was evaluated using luciferase as a reporter protein.
Results showed that high-pressure cell disruption at 30 KPSIG yielded the most efficient lysis and the highest protein concentrations without the use of chemical inhibitors that might impair protein synthesis. Cell extracts from P. pastoris strains engineered for ribosome overexpression achieved up to a 3.4-fold increase in protein yield compared to wild-type strains. The main takeaway is that high-pressure cell disruption combined with strain engineering significantly enhances protein extraction and production efficiency in P. pastoris CFPS systems.
Conclusion on the Role of Constant Systems Cell Disruption equipment:
Constant Systems Equipment played a pivotal role in this research by providing the high-pressure cell disruption necessary to achieve efficient lysis of Pichia pastoris cells. This equipment enabled the researchers to obtain high-quality cell extracts with minimal unlysed cells, which was crucial for maximizing protein yield and performance in subsequent cell-free protein synthesis experiments. Its reliable and consistent cell disruption capabilities were fundamental to the successful implementation of the CFPS platform, providing a robust foundation for therapeutic protein production.
