(129b) Yeast Ribosome Display for High-Throughput Selection of Cap-Independent Translation Regulatory Elements Impacting Both in Vitro and in Vivo Protein Expression

Rui Ganand Michael C. Jewett

Here we report a yeast ribosome display method. This in vitro selection and evolution method, based on a cell-free protein synthesis (CFPS) system derived from Saccharomyces cerevisiae extracts, is able to enrich a target gene from a model library by 100-fold per round of selection. To demonstrate utility of the technology, and in order to improve protein synthesis in the S. cerevisiae CFPS system, we successfully evolved a 5’ end untranslated region (5-UTR) of a reporter protein containing 15 randomized nucleotides in front of the start codon. After 4-rounds of selection, the most beneficial mutant showed a 13-fold improvement in protein synthesis as compared to the starting library, which is mainly due to enhanced cap-independent translation initiation (CITI) efficiency of the selected 5-UTR sequence. By repeating the beneficial short sequences in tandem, protein yield was further improved by ~3-fold in CFPS. Additionally, the sequence information of 24 5-UTR mutants with various CITI activities was collected and the determinants of activity were analyzed. We found that the CITI activity is more dependent on the A/T content (R² = 0.50) than on the free energy (R²=0.19) of the 5-UTR sequence. Finally, twelve 5-UTR mutants were cloned into an in vivo yeast expression vector and tested for protein yield. Surprisingly, these CITI sequences were able to tune protein expression in vivo across a 20-fold range in expression, similar to in vitro results. Thus, the yeast ribosome display method was used to identify robust and reliable genetic elements in vitro prior to putting them into a host. Looking forward, the yeast ribosome display method holds promise for evolving libraries of proteins (e.g., antibodies) as well as DNA regulatory parts (e.g., promoter function, terminator strength) for protein engineering and synthetic biology.