New method for creating human in vitro translation lysates

Graphical overview of the steps for the production of translation-competent lysates using double centrifugation.

The Mühlemann lab developed a new experimental approach to develop in vitro translation lysates from human cells using dual centrifugation, which allows for reproducible lysis conditions without the usage of detergents. Their method has been published in a publication in RNA Biology " Production of human translation-competent 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. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. 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. Reduction of the phosphorylation state of eIF2a using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.

Read the Publication in RNA Biology (Open Acess)

Website Mühlemann Lab

Abstract, figure and title from Gurzeler et al. (2021) RNA Biology published under a CC BY 4.0 license.