TY - JOUR T1 - Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system JF - bioRxiv DO - 10.1101/099838 SP - 099838 AU - Jun Li AU - Chi Zhang AU - Poyi Huang AU - Erkin Kuru AU - Eliot T. C. Forster-Benson AU - Taibo Li AU - George M. Church Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/01/12/099838.abstract N2 - Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to in vitro protein synthesis. Here we show that compared to the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces ~4-fold more total protein. However the majority of these polypeptides are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ~2/3rd of that measured using the S30 crude extract system. Both of the two systems suffer from low ribosome recycling efficiency when translating proteins from 90 kD to 220 kD. A systematic fed-batch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination prior to energy depletion increased Fluc protein yield by ~1.5 to ~2-fold, while accumulation of inorganic phosphate contributes to reaction termination. Additionally, while adding EF-P to PURE reduced total protein translated, it also increased the fraction of active product and reduced partial translated product probably by slowing down the translation process. Finally, ArfA, rather than YaeJ or PrfH, helped reduce ribosome stalling when translating Fluc and improved system productivity in a template-dependent fashion. ER -