Open AccessCell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems
Author(s) -
Baptiste Panthu,
Théophile Ohlmann,
Johan Perrier,
Uwe Schlattner,
Pierre Jalinot,
Bénédicte Elena-Herrmann,
Gilles Rautureau
Publication year - 2017
Publication title -
acs synthetic biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.156
H-Index - 66
ISSN - 2161-5063
DOI - 10.1021/acssynbio.7b00280
Subject(s) - ribosome , protein biosynthesis , cell free protein synthesis , ribosomal protein , lysis , translation (biology) , biochemistry , ribosomal rna , biology , chemistry , biophysics , microbiology and biotechnology , messenger rna , rna , gene
A counterintuitive 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 has an upper limit at native ribosome concentration and that lower amounts of the 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.