Efficient cell-free translation from diverse human cell types

Abstract

MotivationThe difficulties in producing cell-free translation systems from different cell types limit the ability to study regulatory mechanisms that depend on different biological contexts. Developing systems tailored to diverse cell types would be instrumental in investigating cell-type-specific translational control, co- and post-translational modifications, and viral manipulation strategies. Our method addresses this gap by providing a scalable and adaptable solution for producing high-quality lysates that reflect the specific needs of different cell types. SummaryCell-free translation systems are indispensable for studying protein synthesis, enabling researchers to explore translational regulation across different cell types. Here, we present a scalable method for preparing translation-competent lysates from a range of frequently used human cell lines using dual centrifugation. We optimized lysis conditions for adherent and suspension cells, producing high-quality lysates from HEK-293 (adherent and in suspension), HeLa, SH-SY5Y, and U2OS cells. Our results demonstrate that cell-specific factors influence translation efficiency, with adherent HeLa cells showing the highest activity. We also observed that sensitivity to lysis conditions varies between cell lines, underscoring the importance of fine-tuning parameters for efficient protein production. Our method provides a robust and adaptable approach for generating cell-type-specific lysates, broadening the application of in vitro translation systems in studying translational mechanisms.