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A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems.

ABSTRACT: Cell-free protein synthesis (CFPS) systems were designed to produce proteins with a minimal set of purified components, thus offering the possibility to follow translation as well as protein folding. In order to characterize the performance of the ribosomes in such a system, it is crucial to separately quantify the two main components of productivity, namely the fraction of active ribosomes and the number of synthesizing cycles. Here, we provide a direct and highly reliable measure of ribosomal activity in any given CFPS system, introducing an enhanced-arrest peptide variant. We observe an almost complete stalling of ribosomes that produce GFPem (~95%), as determined by common centrifugation techniques and fluorescence correlation spectroscopy (FCS). Moreover, we thoroughly study the effect of different ribosomal modifications independently on activity and number of synthesizing cycles. Finally, employing two-colour coincidence detection and two-colour colocalisation microscopy, we demonstrate real-time access to key productivity parameters with minimal sample consumption on a single ribosome level.

SUBMITTER: Kempf N

PROVIDER: S-EPMC5402277 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems.

Kempf Noémie N Remes Cristina C Ledesch Ralph R Züchner Tina T Höfig Henning H Ritter Ilona I Katranidis Alexandros A Fitter Jörg J

Scientific reports 20170424

Cell-free protein synthesis (CFPS) systems were designed to produce proteins with a minimal set of purified components, thus offering the possibility to follow translation as well as protein folding. In order to characterize the performance of the ribosomes in such a system, it is crucial to separately quantify the two main components of productivity, namely the fraction of active ribosomes and the number of synthesizing cycles. Here, we provide a direct and highly reliable measure of ribosomal ...[more]

PMID: 28436469

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Project description:We have developed and characterized translationally active cell-free systems from Artemia embryos at different developmental times. The optimized lysates from 16 h-developed embryos incorporated radiolabelled amino acids into polypeptides for up to 120 min. The polypeptides synthesized ranged in Mr from 150,000 to 10,000, suggesting that the endogenous mRNA was capable of directing the synthesis of complete polypeptides. Similar results were obtained by using lysates from early developmental stages; even the cell-free system prepared from 1 h-developed embryos was partially active in protein synthesis. Furthermore, all these lysates were capable of re-initiation, as demonstrated by inhibition of initiation with the inhibitors edeine and 7-methylguanosine 5'-triphosphate. Because we found no endogenous protein-synthetic activity in the corresponding lysates from undeveloped embryos, we have used cell-free translation systems from 0 h- and 16 h-developed Artemia embryos to analyse the mechanisms limiting protein synthesis at very early developmental stages. Undeveloped-embryo lysates supplemented with nuclease-treated reticulocyte lysate were capable of translating endogenous mRNAs to give products with a wide spectrum of Mr values, but lysates of 16 h-developed embryos supplemented in this way were not further stimulated. The nuclease-treated lysate appeared to be unnecessary 5 h after resumption of development. These results suggested that a component(s) limiting translation in the undeveloped-embryo lysate was provided by the nuclease-treated reticulocyte lysate, and that this component(s) no longer limited protein synthesis after development. In view of these results, partially fractionated reticulocyte lysates were tested for restoration of protein-synthetic activity in the undeveloped embryo lysate. A high-salt ribosomal wash devoid of ribosomal subunits, which is considered a crude polypeptide-initiation-factor preparation, also restored translation activity in the undeveloped embryo lysate and made it capable of directing the synthesis of both endogenous mRNAs and exogenous (globin) mRNA.

Project description:Cell-free bioproduction systems represent a promising alternative to classical microbial fermentation processes to synthesize value-added products from biological feedstocks. An essential step for establishing cell-free production systems is the identification of suitable metabolic modules with defined properties. Here we present MEMO, a novel computational approach to find smallest metabolic modules with specified stoichiometric and thermodynamic constraints supporting the design of cell-free systems in various regards. In particular, one key challenge for a sustained operation of cell-free systems is the regeneration of utilized cofactors (such as ATP and NAD(P)H). Given a production pathway with certain cofactor requirements, MEMO can be used to compute smallest regeneration modules that recover these cofactors with required stoichiometries. MEMO incorporates the stoichiometric and thermodynamic constraints in a single mixed-integer linear program, which can then be solved to find smallest suitable modules from a given reaction database. We illustrate the applicability of MEMO by calculating regeneration modules for the recently published synthetic CETCH cycle for in vitro carbon dioxide fixation. We demonstrate that MEMO is very flexible in taking into account the diverse constraints of the CETCH cycle (e.g., regeneration of 1 ATP, 4 NADPH and of 1 acetyl-group without net production of CO2 and with permitted side production of malate) and is able to determine multiple solutions in reasonable time in two large reaction databases (MetaCyc and BiGG). The most promising regeneration modules found utilize glycerol as substrate and require only 8 enzymatic steps. It is also shown that some of these modules are robust against spontaneous loss of cofactors (e.g., oxidation of NAD(P)H or hydrolysis of ATP). Furthermore, we demonstrate that MEMO can also find cell-free production systems with integrated product synthesis and cofactor regeneration. Overall, MEMO provides a powerful method for finding metabolic modules and for designing cell-free production systems as one particular application.

Dataset Information

A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems.

Publications

A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems.

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