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  4. Towards Self-regeneration: Exploring the Limits of Protein Synthesis in the Protein Synthesis Using Recombinant Elements (PURE) Cell-free Transcription-Translation System
 
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research article

Towards Self-regeneration: Exploring the Limits of Protein Synthesis in the Protein Synthesis Using Recombinant Elements (PURE) Cell-free Transcription-Translation System

Ganesh, Ragunathan B.  
•
Maerkl, Sebastian J.  
July 27, 2024
ACS Synthetic Biology

Self-regeneration is a key function of living systems that needs to be recapitulated in vitro to create a living synthetic cell. A major limiting factor for protein self-regeneration in the PURE cell-free transcription-translation system is its high protein concentration, which far exceeds the system's protein synthesis rate. Here, we were able to drastically reduce the nonribosomal PURE protein concentration up to 97.3% while increasing protein synthesis efficiency. Although crowding agents were not effective in the original PURE formulation, we found that in highly dilute PURE formulations, addition of 6% dextran considerably increased protein synthesis rate and total protein yield. These new PURE formulations will be useful for many cell-free synthetic biology applications, and we estimate that PURE can now support the complete self-regeneration of all 36 nonribosomal proteins, which is a critical step toward the development of a universal biochemical constructor and living synthetic cell.

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Type
research article
DOI
10.1021/acssynbio.4c00304
Web of Science ID

WOS:001279688200001

PubMed ID

39066734

Author(s)
Ganesh, Ragunathan B.  
•
Maerkl, Sebastian J.  
Date Issued

2024-07-27

Publisher

AMER CHEMICAL SOC

Published in
ACS Synthetic Biology
Volume

13

Issue

8

Start page

2555

End page

2566

Subjects

synthetic biology

•

PURE system

•

cell-free transcriptionand translation

•

crowding agents

•

protein regeneration

Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
LBNC  
FunderFunding(s)Grant NumberGrant URL

European Research Council (ERC)

723106

Swiss National Science Foundation (SNSF)

514558

Swiss National Science Foundation MINT grant

514337

Available on Infoscience
January 30, 2025
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/245914
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