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Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

Jun Li, Chi Zhang, Poyi Huang, Erkin Kuru, Eliot T. C. Forster-Benson, Taibo Li, George M. Church
doi: https://doi.org/10.1101/099838
Jun Li
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
2Wyss Harvard Institute of Biologically Inspired Engineering, 3 Blackfan Circle, Boston, MA 02115, USA.
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  • For correspondence: junlieugene@gmail.com gchurch@genetics.med.harvard.edu
Chi Zhang
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
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Poyi Huang
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
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Erkin Kuru
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
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Eliot T. C. Forster-Benson
3Ravenwood High School, 1724 Wilson Pike, Brentwood, TN 37027, USA.
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Taibo Li
4Department of Electrical Engineering and Computer Science, Massachusetts of Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
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George M. Church
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
2Wyss Harvard Institute of Biologically Inspired Engineering, 3 Blackfan Circle, Boston, MA 02115, USA.
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  • For correspondence: junlieugene@gmail.com gchurch@genetics.med.harvard.edu
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ABSTRACT

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.

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Posted January 12, 2017.
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Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system
Jun Li, Chi Zhang, Poyi Huang, Erkin Kuru, Eliot T. C. Forster-Benson, Taibo Li, George M. Church
bioRxiv 099838; doi: https://doi.org/10.1101/099838
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Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system
Jun Li, Chi Zhang, Poyi Huang, Erkin Kuru, Eliot T. C. Forster-Benson, Taibo Li, George M. Church
bioRxiv 099838; doi: https://doi.org/10.1101/099838

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