Dataset Information

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

ABSTRACT: 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 stalling when translating Fluc and improved system productivity in a template-dependent fashion.

SUBMITTER: Li J

PROVIDER: S-EPMC5501384 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Li Jun J Zhang Chi C Huang Poyi P Kuru Erkin E Forster-Benson Eliot T C ETC Li Taibo T Church George M GM

Translation (Austin, Tex.) 20170509 1

Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to <i>in vitro</i> protein synthesis. Here we show that compared with the commercial S30 crude extract based RTS 100 <i>E. coli</i> 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 sign ...[more]

PMID: 28702280

Similar Datasets

Project description:1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH(4)Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH(4)Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH(4)Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH(4)Cl. 7. NH(4)Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH(4)Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.

Project description:BACKGROUND:Fish skin microbiomes are rarely studied in inland water systems, in spite of their importance for fish health and ecology. This is mainly because fish species distribution often covaries with other biotic and abiotic factors, complicating the study design. We tackled this issue in the northern part of the Jordan River system, in which a few fish species geographically overlap, across steep gradients of water temperature and salinity. RESULTS:Using 16S rRNA metabarcoding, we studied the water properties that shape the skin bacterial communities, and their interaction with fish taxonomy. To better characterise the indigenous skin community, we excluded bacteria that were equally abundant in the skin samples and in the water samples, from our analysis of the skin samples. With this in mind, we found alpha diversity of the skin communities to be stable across sites, but higher in benthic loaches, compared to other fish. Beta diversity was found to be different among sites and to weakly covary with the dissolved oxygen, when treated skin communities were considered. In contrast, water temperature and conductivity were strong factors explaining beta diversity in the untreated skin communities. Beta diversity differences between co-occurring fish species emerged only for the treated skin communities. Metagenomics predictions highlighted the microbiome functional implications of excluding the water community contamination from the fish skin communities. Finally, we found that human-induced eutrophication promotes dysbiosis of the fish skin community, with signatures relating to fish health. CONCLUSIONS:Consideration of the background water microbiome when studying fish skin microbiomes, across varying fish species and water properties, exposes patterns otherwise undetected and highlight among-fish-species differences. We suggest that sporadic nutrient pollution events, otherwise undetected, drive fish skin communities to dysbiosis. This finding is in line with a recent study, showing that biofilms capture sporadic pollution events, undetectable by interspersed water monitoring. Video abstract.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets