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

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 ~2/3 rd 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 k D to 224 k D. 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 stallingmore » when translating Fluc and improved system productivity in a template-dependent fashion.« less
 [1] ;  [2] ;  [2] ;  [2] ;  [3] ; ORCiD logo [4] ;  [1]
  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
  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
Publication Date:
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 5; Journal Issue: 1; Related Information: Supplementary information document is available on the journal website; Journal ID: ISSN 2169-0731
Research Org:
Harvard Univ., Boston, MA (United States). Harvard Medical School
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; cell-free protein synthesis; ribosome stalling; translation
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