Production of translation-competent human cell lysates using dual centrifugation
Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions.
With the funding that was acquired thanks to the UniBern Forschungsstiftungs grant we obtained a dual centrifugation device that allows for detergent-free cell lysis under controlled mechanical forces (published in Gurzeler et al., RNA biol., 2022).
We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Using the derived lysates, we contributed to elucidating the role of Nsp1, a potent virulent factor produced during the early steps of infection by SARS-CoV-2 in human cells (Schubert, Karousis et al., Nature Struct. Mol. Biol, 2020) in collaboration with the group of Nenad Ban.
Additionally, we routinely use the technique now for immunoprecipitation experiments or for structural studies and we explore the potential for using it as a method of cell fractionation. The next goal is to develop a screening platform for human translation inhibitors based on in vitro translation. Therefore, the acquisition of this equipment led to the development of new exciting projects in the field of human translation.
Evangelos D. Karousis, PhD
Dept. of Chemistry, Biochemistry and Pharmaceutical Sciences
Die Projektförderung wurde ermöglicht durch einen Beitrag des BEKB Förderfonds
