Ribosomal Protein S1 Improves the Protein Yield of an In Vitro Reconstituted Cell-Free Translation System

This is the authors' abstract. We don't add key points for this paper.

Cell-free expression systems, such as the highly purified in vitro reconstituted PURExpress, hold great promise for engineering biological and life-similar systems by exploiting the ability to perform transcription and translation (TX–TL) outside the constraints of living cells, including for example the expression of recombinant proteins that are difficult or toxic to produce in vivo . Currently, the scope of applications utilizing purified reconstituted TX–TL systems is challenged by poor system performance resulting from limitations in the ribosome and ribosome-associated processes, leading to low protein yields. Because of the transient nature of ribosomal protein S1’s interaction with the ribosome, the ribosomes in a reconstituted translation system contain varying amounts of S1, potentially impacting translation initiation and the recruitment of mRNA to the 30S ribosomal subunit. Here we report that by being supplemented with purified recombinant S1 the protein yields can be doubled when using a commercial in vitro reconstituted TX–TL system. We hypothesize that the addition of S1 increases the fraction of functional ribosomes available in the in vitro reaction. Improved yields are shown for different reporter proteins (EYFP, sfGFP, and mRFP) and in different 5′UTR contexts (strong, medium, and weak ribosome binding site), including the expression of a highly structured RNA (PSIV IRES). Overall, fine-tuning the S1 concentration provides a previously overlooked venue to increase protein yield by targeting ribosome composition and translation initiation.

Enables quantitative prediction of PURE-based TX-TL; leaves open validation across proteins and conditions.