A highly optimized human in-vitro translation system
A new method paper published by the Ban group (IMBB) in “Cell Reports Methods” describes the production and use of an improved Human In-vitro Translation System (HITS), that is capable of highly efficient and regulated ribosomal protein synthesis in a cell-free environment.
To study cellular protein synthesis, which is catalyzed by ribosomes, in-vitro translation systems have been an important tool for many decades. They allow direct manipulation of the process, for example programmed translation from synthetic mRNAs, and are easily compatible with downstream structural biology techniques, most importantly cryo-electron microscopy. Until now, the most popular in-vitro translation system was one prepared from rabbit blood cells, which for many reasons is a suboptimal system when studying the regulation of mammalian translation. In contrast, Human In-vitro Translation Systems (HITSs) have been around for 5 decades but always had the reputation of being difficult to prepare and rather inefficient.
The work by Bothe & Ban revisited the problem of preparing a highly active HITS from cultured human cells. Through systematic optimization of components, they were able to simplify the system, removing three components that were ubiquitously used in HITSs until now. Meanwhile, through screenings the optimal conditions for translation were found, resulting in a more than 10-fold increase in translation efficiency. The new HITS presented by the Ban group is easy to prepare as well as easier to use and cheaper than commercial alternatives.
Through biochemical assays, the researchers showed that hallmarks of cellular translation regulation are recapitulated in their HITS, proving that it will be a useful tool for studying the intricacies of human translation regulation.
Link to the paper in external page “Cell Reports Methods”.