Abstract
A counter-intuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity is ceiled at native ribosome concentration and that lower amounts of ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.
PMID: 28915016 [PubMed - as supplied by publisher]
[NMR paper] Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.
Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.
Related Articles Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.
Pharmaceuticals (Basel). 2017 Apr 15;10(2):
Authors: Takechi-Haraya Y, Aki K, Tohyama Y, Harano Y, Kawakami T, Saito H, Okamura E
Abstract
Glycosaminoglycans (GAGs), which are covalently-linked membrane proteins at the cell...
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Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression
Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/0/bichaw.ahead-of-print/acs.biochem.6b00488/20160721/images/medium/bi-2016-00488z_0007.gif
Biochemistry
DOI: 10.1021/acs.biochem.6b00488
http://feeds.feedburner.com/~ff/acs/bichaw?d=yIl2AUoC8zA
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07-22-2016 01:34 AM
[NMR paper] Real-Time Monitoring of Cancer Cell Metabolism and Effects of an Anticancer Agent using 2D In-Cell NMR Spectroscopy.
Real-Time Monitoring of Cancer Cell Metabolism and Effects of an Anticancer Agent using 2D In-Cell NMR Spectroscopy.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--media.wiley.com-assets-2250-98-WileyOnlineLibrary_FullTextOnline_120x27.gif Related Articles Real-Time Monitoring of Cancer Cell Metabolism and Effects of an Anticancer Agent using 2D In-Cell NMR Spectroscopy.
Angew Chem Int Ed Engl. 2015 Mar 5;
Authors: Wen H, An YJ, Xu WJ, Kang KW, Park S
Abstract
Altered metabolism is a critical part of...
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03-11-2015 09:59 PM
[NMR paper] Real-time NMR monitoring of protein-folding kinetics by a recycle flow system for temperature jump.
Real-time NMR monitoring of protein-folding kinetics by a recycle flow system for temperature jump.
Real-time NMR monitoring of protein-folding kinetics by a recycle flow system for temperature jump.
Anal Chem. 2013 Sep 12;
Authors: Yamasaki K, Obara Y, Hasegawa M, Tanaka H, Yamasaki T, Wakuda T, Okada M, Kohzuma T
Abstract
An NMR method was developed that allows for real-time monitoring of reactions (on the order of seconds) induced by temperature jump. In a recycle flow system, heating and cooling baths were integrated, with the latter...
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09-14-2013 03:02 PM
[NMR paper] Real-time kinetics of high-mobility group box 1 oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
Real-time kinetics of high-mobility group box 1 oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
Real-time kinetics of high-mobility group box 1 oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
J Biol Chem. 2013 Feb 27;
Authors: Zandarashvili L, Sahu D, Lee K, Lee YS, Singh P, Rajarathnam K, Iwahara J
Abstract
Some extracellular proteins are initially secreted in the reduced form via a non-canonical pathway bypassing the endoplasmic reticulum, and become oxidized in the...
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03-01-2013 09:57 PM
Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR.
Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR.
Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR.
Nat Chem Biol. 2011 Mar 20;
Authors: Ullrich SJ, Hellmich UA, Ullrich S, Glaubitz C
The simultaneous observation of interdependent reactions within different phases as catalyzed by membrane-bound enzymes is still a challenging task. One such enzyme, the Escherichia coli integral membrane protein diacylglycerol kinase (DGK), is a key player in lipid regulation. It catalyzes the...
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03-23-2011 05:41 PM
[NMR paper] Folding kinetics of the SH3 domain of PI3 kinase by real-time NMR combined with optic
Folding kinetics of the SH3 domain of PI3 kinase by real-time NMR combined with optical spectroscopy.
Related Articles Folding kinetics of the SH3 domain of PI3 kinase by real-time NMR combined with optical spectroscopy.
J Mol Biol. 1998 Feb 27;276(3):657-67
Authors: Guijarro JI, Morton CJ, Plaxco KW, Campbell ID, Dobson CM
The refolding kinetics of the chemically denatured SH3 domain of phosphatidylinositol 3'-kinase (PI3-SH3) have been monitored by real-time one-dimensional 1H NMR coupled with a variety of other biophysical techniques. These...
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11-17-2010 11:06 PM
Cell-free protein synthesis of perdeuterated proteins for NMR studies
Cell-free protein synthesis of perdeuterated proteins for NMR studies
Touraj Etezady-Esfarjani, Sebastian Hiller, Cristina Villalba and Kurt Wüthrich
Journal of Biomolecular NMR; 2007; 39(3); pp 229-238
Abstract:
Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins...
Cell-free protein synthesis enhancement from real-time NMR metabolite kinetics: redirecting energy fluxes in hybrid RRL systems.
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