Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems

Hansen, Maike M. K.; Rosquelles, Marta Ventosa; Yelleswarapu, Maaruthy; Maas, Roel; Vugt-Jonker, Aafke J. van; Heus, Hans A.; Huck, Wilhelm T. S.

doi:10.1021/acssynbio.6b00010

Cited by 17 publications

(26 citation statements)

References 39 publications

(87 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Since the sequences of these two genes greatly differed (48% identity), it seemed likely that the folding of the mRNA strongly influenced protein expression, as previously noted. 25,26 No correlation was found between gene expression and the position of the base pairing interactions within the RBS nor the composition of the non-base pairing positions within the RBS. Taken together, RBS sequences significantly impacted protein expression, as expected, but the effects were not consistent between different genetic sequences, likely reflecting the influences of competing intramolecular base pairing interactions.…”

Section: Ribosome Binding Sites Strongly But Unreliably Control Gene mentioning

confidence: 96%

“…16 A likely explanation for this discrepancy is the decoupling of RNA and protein synthesis when using T7 RNA polymerase and E. coli ribosomes. 1,16,25 Perhaps substituting T7 RNA polymerase with E. coli RNA polymerase, as in ePURE, 34 would allow for proper coupling and thus the ability to predictably control protein output through the activity of the transcriptional promoter.…”

Section: A Better Understanding Of the Influence Of Rna Folding On Trmentioning

confidence: 99%

See 1 more Smart Citation

Cell-free translation is more variable than transcription

Chizzolini

Forlin

Mansy

2016

Preprint

View full text Add to dashboard Cite

Although RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, a series of ribosome binding sites (RBS) was investigated. While RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including GC content.The complexity of protein synthesis in comparison to RNA synthesis was observed by the higher degree of variability associated with protein expression. This variability was also observed in an E. coli cell extractbased system. However, the coefficient of variation was larger with E. coli RNA polymerase than with T7 RNA polymerase, consistent with the increased complexity of E. coli RNA polymerase.

show abstract

Section: Ribosome Binding Sites Strongly But Unreliably Control Gene mentioning

confidence: 96%

Section: A Better Understanding Of the Influence Of Rna Folding On Trmentioning

confidence: 99%

Cell-free translation is more variable than transcription

Chizzolini

Forlin

Mansy

2016

Preprint

View full text Add to dashboard Cite

show abstract

“…[24] Allerdings entkoppelt schon der Toehold-Switch-Riboregulator selbst diese beiden Prozesse -Ribosomen kçnnen nicht direkt an neugebildete mRNAb inden -, weswegen in unserem Fall die Expressionsrate wohl einfach infolge des geringeren Reaktionsvolumens reduziert ist. Es wurde zuvor beschrieben, dass die Tr ennung von Tr anskription und Tr anslation die Effizienz von ZFPE-Reaktionen reduziert.…”

Section: Angewandte Chemieunclassified

Künstliche, gelbasierte Organellen für die räumliche Organisation von zellfreien Genexpressionsreaktionen

Aufinger

Simmel

2018

Angewandte Chemie

View full text Add to dashboard Cite

Gelbasierte,k ünstliche Organellen wurden entwickelt, die die sequenzspezifische und programmierbare Lokalisation zellfreier Transkriptions-und Translationsreaktionen innerhalb eines künstlichen zellulären Systems ermçglichen. Dazu wurden Agarose-Mikrogele genutzt, die kovalent mit DNA-Templaten modifiziert wurden, die fürverschiedene Funktionen codieren, und diese wiederum in Emulsionstrçpfchen eingeschlossen. VonT ranskriptionsorganellent ranskribierte RNA-Signale kçnnen spezifisch,d urchH ybridisierung mit entsprechenden DNA-Adressmolekülen, von Zielorganellen eingefangen werden. mRNA-Moleküle mit Toehold-Switch-Riboregulatoren, die von Transkriptionsorganellen produziert werden, werden nur in Translationsorganellen exprimiert, die den entsprechenden DNA-Trigger fürd en Riboregulator enthalten. Die räumlicheB eschränkung von Transkription und Translation auf getrennte Organellen ähnelt damit der Genexpression in eukaryotischen Zellen. Die Kombination kommunizierender Gelkügelchen mit spezialisierten Funktionen erçffnet die Mçglichkeit der Programmierung künstlicher zellulärer Systeme auf Organell-Ebene.

show abstract

“…Firstly, since there is no need to support cellular metabolism; all of the cellular resources can be efficiently directed toward the production of a single protein [5]. Although the coupled (i.e., combining both transcription and translation processes) CF system has been proven to be more efficient than the uncoupled one [6], it is still possible to use mRNA or PCR fragments as the matrices escaping genetic engineering and cloning procedures [7]. Taken together, this makes CF technology a reliable and fast way to obtain a high yield of the desired protein.…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

Smolskaya

Logashina

Andreev

2020

IJMS

View full text Add to dashboard Cite

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.

show abstract

Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems

Cited by 17 publications

References 39 publications

Cell-free translation is more variable than transcription

Cell-free translation is more variable than transcription

Künstliche, gelbasierte Organellen für die räumliche Organisation von zellfreien Genexpressionsreaktionen

Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

Contact Info

Product

Resources

About