Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell

2016 ◽  
Vol 22 (2) ◽  
pp. 185-195 ◽  
Author(s):  
Filippo Caschera ◽  
Vincent Noireaux
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Coli Cell ◽  
Free Expression ◽  
Genetic Circuit ◽  
Translation System ◽  
Minimal Cell ◽  
E Coli ◽  
Synthetic Cell

Cell-free expression is a technology used to synthesize minimal biological cells from natural molecular components. We have developed a versatile and powerful all-E. coli cell-free transcription–translation system energized by a robust metabolism, with the far objective of constructing a synthetic cell capable of self-reproduction. Inorganic phosphate (iP), a byproduct of protein synthesis, is recycled through polysugar catabolism to regenerate ATP (adenosine triphosphate) and thus supports long-lived and highly efficient protein synthesis in vitro. This cell-free TX-TL system is encapsulated into cell-sized unilamellar liposomes to express synthetic DNA programs. In this work, we study the compartmentalization of cell-free TX-TL reactions, one of the aspects of minimal cell module integration. We analyze the signals of various liposome populations by fluorescence microscopy for one and for two reporter genes, and for an inducible genetic circuit. We show that small nutrient molecules and proteins are encapsulated uniformly in the liposomes with small fluctuations. However, cell-free expression displays large fluctuations in signals among the same population, which are due to heterogeneous encapsulation of the DNA template. Consequently, the correlations of gene expression with the compartment dimension are difficult to predict accurately. Larger vesicles can have either low or high protein yields.

scholarly journals Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro

2018 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg ◽  
Hui Ge
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Therapeutic Index ◽  
Antiviral Protein ◽  
E Coli ◽  
Protein Isoform ◽  
Antiviral Proteins ◽  
A Chain

AbstractRicin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with 50% protein synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and protein synthesis inhibition activity assayed. Results showed that the optimized protein RTA mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on protein synthesis inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infection.

scholarly journals A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA in an All-E. Coli Cell-Free Expression System

2020 ◽  
Author(s):  
Emanuel Worst ◽  
oemer Kurt ◽  
Marc Finkler ◽  
Marc Schenkelberger ◽  
Vincent Noireaux ◽  
...  
Keyword(s):  
Expression System ◽  
Regulatory Region ◽  
Phase Variation ◽  
Coli Strain ◽  
Coli Cell ◽  
Free Expression ◽  
E Coli ◽  
Regulatory Dna ◽  
Cell Free Expression System

<p>Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary, alternate DNA methylation of only two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study the function of pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. Based on our observations we suggest that Lrp and the conformation of the self-complementary regulatory DNA play a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for engineering stable, hereditary and strong expression control based on methylation.</p>

Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system

2008 ◽  
Vol 389 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Stephan Frey ◽  
Martin Haslbeck ◽  
Otmar Hainzl ◽  
Johannes Buchner
Keyword(s):  
Mammalian Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Basic Research ◽  
Medical Diagnostics ◽  
Free Expression ◽  
Translation System ◽  
Functional Antibodies ◽  
The Stability

Abstract Antibodies are an important component of the immune system of higher eukaryotes. Furthermore, they are effective tools in basic research, medical diagnostics and therapy. Recombinant expression of these heterotetrameric, disulfide-bridged proteins is usually performed in mammalian cells. Here, we describe the cell-free expression of a mouse monoclonal antibody, MAK33, in a coupled transcription/translation system, based on an Escherichia coli lysate. Both the heavy and the light chain can be produced efficiently in this setup. However, they fail to form functional antibodies. With a view to overcome folding and oxidation defects, we supplemented the system with the oxidoreductases PDI (protein disulfide isomerase) and DsbC and the ER-specific chaperones Grp94 and BiP; furthermore, we optimized the redox conditions. We found that functional antibodies can only be obtained in the presence of an oxidoreductase. In contrast, the addition of Grp94 and/or BiP had no influence on the productive folding reaction. The comparison of the antibody expressed in vitro with MAK33 expressed in cell culture showed that the in vitro expressed antibody is correctly assembled, disulfide-bridged and shows identical antigen affinity. The stability of the in vitro expressed non-glycosylated IgG is comparable to that of the authentic antibody.

Anaerobic Conditioning of E. coli Cell Lysate for Enhanced In Vitro Protein Synthesis

2021 ◽  
Vol 10 (4) ◽  
pp. 716-723
Author(s):  
By Denis Tamiev ◽  
Jared L. Dopp ◽  
Nigel F. Reuel
Keyword(s):  
Protein Synthesis ◽  
Coli Cell ◽  
In Vitro Protein Synthesis ◽  
E Coli ◽  
Cell Lysate ◽  
Vitro Protein

scholarly journals A Crude Extract Preparation and Optimization from a Genomically Engineered Escherichia coli for the Cell-Free Protein Synthesis System: Practical Laboratory Guideline

2019 ◽  
Vol 2 (3) ◽  
pp. 68 ◽  
Author(s):  
Kim ◽  
Copeland ◽  
Padumane ◽  
Kwon
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Optimization Procedure ◽  
Cell Extract ◽  
Coli Cell ◽  
Free Protein ◽  
E Coli ◽  
Highly Active ◽  
Cell Free Protein Synthesis

With the advancement of synthetic biology, the cell-free protein synthesis (CFPS) system has been receiving the spotlight as a versatile toolkit for engineering natural and unnatural biological systems. The CFPS system reassembles the materials necessary for transcription and translation and recreates the in vitro protein synthesis environment by escaping a physical living boundary. The cell extract plays an essential role in this in vitro format. Here, we propose a practical protocol and method for Escherichia coli-derived cell extract preparation and optimization, which can be easily applied to both commercially available and genomically engineered E. coli strains. The protocol includes: (1) The preparation step for cell growth and harvest, (2) the thorough step-by-step procedures for E. coli cell extract preparation including the cell wash and lysis, centrifugation, runoff reaction, and dialysis, (3) the preparation for the CFPS reaction components and, (4) the quantification of cell extract and cell-free synthesized protein. We anticipate that the protocol in this research will provide a simple preparation and optimization procedure of a highly active E. coli cell extract.

scholarly journals Incompatibility of DFHBI based fluorescent RNA aptamers with particular commercial cell-free expression systems

2021 ◽  
Author(s):  
Alexander J Speakman ◽  
Katherine E Dunn
Keyword(s):  
Expression System ◽  
In Vitro Transcription ◽  
Rna Aptamers ◽  
Free Expression ◽  
Reaction Products ◽  
Expression Systems ◽  
E Coli ◽  
Cell Free Expression System

Fluorescent RNA aptamers are an increasingly used tool for quantifying transcription and for visualising RNA interactions, both in vitro and in vivo. However when tested in the commercially available, E. coli extract based Expressway™ cell-free expression system, no fluorescence is detected. The same experimental setup is shown to successfully produce fluorescent RNA aptamers when tested in another buffer designed for in vitro transcription, and RNA purification of the Expressway™ reaction products show that transcription does occur, but does not result in a fluorescent product. In this paper we demonstrate the incompatibility of a narrow selection of RNA aptamers in one particular cell-free expression system, and consider that similar issues may arise with other cell-free expression systems, RNA aptamers, and their corresponding fluorophores.

scholarly journals A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA in an All-E. Coli Cell-Free Expression System

2020 ◽  
Author(s):  
Emanuel Worst ◽  
oemer Kurt ◽  
Marc Finkler ◽  
Marc Schenkelberger ◽  
Vincent Noireaux ◽  
...  
Keyword(s):  
Expression System ◽  
Regulatory Region ◽  
Phase Variation ◽  
Coli Strain ◽  
Coli Cell ◽  
Free Expression ◽  
E Coli ◽  
Regulatory Dna ◽  
Cell Free Expression System

<p>Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary, alternate DNA methylation of only two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study the function of pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. Based on our observations we suggest that Lrp and the conformation of the self-complementary regulatory DNA play a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for engineering stable, hereditary and strong expression control based on methylation.</p>

Sign in / Sign up

Export Citation Format

Share Document