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

Journal Article · · Translation
 [1];  [2];  [2];  [2];  [3];  [4];  [5]
  1. Harvard Univ., Boston, MA (United States). Harvard Medical School, Dept. of Genetics; Harvard Univ., Boston, MA (United States). Wyss-Harvard Inst. of Biologically Inspired Engineering; Department of Genetics, Harvard Medical School
  2. Harvard Univ., Boston, MA (United States). Harvard Medical School, Dept. of Genetics
  3. Ravenwood High School, Brentwood, TN (United States)
  4. Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Electrical Engineering and Computer Science
  5. Harvard Univ., Boston, MA (United States). Harvard Medical School, Dept. of Genetics; Harvard Univ., Boston, MA (United States). Wyss-Harvard Inst. of Biologically Inspired Engineering
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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 with the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces up to ~6-fold full-length proteins. However the majority of polypeptides PURE produces are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ~2/3rd of that measured using the RTS 100 E. coli HY S30 system. Both of the 2 batch systems suffer from low ribosome recycling efficiency when translating proteins from 82 kD to 224 kD. A systematic fed-batch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination before energy depletion increased Fluc protein yield by ~1.5 to ~2-fold, while creatine phosphate and magnesium have synergistic effects when added to the PURE system. Additionally, while adding EF-P to PURE reduced full-length protein translated, it increased the fraction of functional protein and reduced partially translated protein 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.
Research Organization:
Harvard Univ., Boston, MA (United States). Harvard Medical School
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Grant/Contract Number:
FG02-02ER63445
OSTI ID:
1373362
Journal Information:
Translation, Journal Name: Translation Journal Issue: 1 Vol. 5; ISSN 2169-0731
Country of Publication:
United States
Language:
English

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Cited By (7)

Strategies for in vitro engineering of the translation machinery journal November 2019
Bottom-up construction of complex biomolecular systems with cell-free synthetic biology text January 2020
Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems journal December 2019
Anomalous Scaling of Gene Expression in Confined Cell-Free Reactions journal May 2018
Modelling cell-free RNA and protein synthesis with minimal systems journal January 2019
Anomalous Scaling of Gene Expression in Confined Cell-Free Reactions posted_content January 2018
Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems posted_content August 2019

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
cell-free protein synthesis
ribosome stalling
translation