RT Journal Article
SR Electronic
T1 Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 099838
DO 10.1101/099838
A1 Jun Li
A1 Chi Zhang
A1 Poyi Huang
A1 Erkin Kuru
A1 Eliot T. C. Forster-Benson
A1 Taibo Li
A1 George M. Church
YR 2017
UL http://biorxiv.org/content/early/2017/01/12/099838.abstract
AB 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.