scholarly journals Uncovering cell-free protein expression dynamics by a promoter library with diverse strengths

2017 ◽  
Author(s):  
Sabrina Galiñanes Reyes ◽  
Yutetsu Kuruma ◽  
Soichiro Tsuda
Keyword(s):  
Protein Expression ◽  
Systems Approach ◽  
Protein Translation ◽  
Limiting Factors ◽  
T7 Promoter ◽  
Free System ◽  
Free Protein ◽  
Pure System ◽  
E Coli ◽  
Promoter Library

AbstractCell-free protein expression systems have been widely used for synthetic biology and metabolic engineering applications in recent years. Yet little is known about protein expression in the cell-free systems. Here we take a systems approach to uncover underlying dynamics of cell-free protein expression. We construct a set of T7 promoter variants to express proteins at different transcription rates in a reconstituted and E. coli extract-based cell-free systems. We find that the maximum expression level and the rate of protein synthesis as responses to the transcription rate change are different in the two cell-free systems, suggesting they are driven by different expression dynamics. We confirm this by constructing a simple mathematical model for each cell-free system, which well reproduce the experimental results and also identify different limiting factors for better protein expression in the two cell-free systems. In particular, they revealed there is a negative feedback effect in the mRNA-protein translation by the PURE system and also identified different limiting factors for better protein expression in the two cell-free systems.

scholarly journals Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

2020 ◽  
Vol 20 ◽  
pp. 04004
Author(s):  
Ahmad Pandu Satria Wiratama ◽  
Aris Haryanto
Keyword(s):  
Protein Expression ◽  
Newcastle Disease ◽  
Recombinant Plasmid ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
E Coli ◽  
Protein Expression System ◽  
Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

scholarly journals Methods to reduce variability in E. Coli-based cell-free protein expression experiments

2019 ◽  
Vol 4 (4) ◽  
pp. 204-211 ◽  
Author(s):  
Jared L. Dopp ◽  
Yeong Ran Jo ◽  
Nigel F. Reuel
Keyword(s):  
Protein Expression ◽  
Free Protein ◽  
E Coli

scholarly journals The Streptomyces coelicolor genome encodes a type I ribosome-inactivating protein

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 3021-3030 ◽  
Author(s):  
Ana G. Reyes ◽  
Nick Geukens ◽  
Philip Gutschoven ◽  
Stijn De Graeve ◽  
René De Mot ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Streptomyces Coelicolor ◽  
Protein Translation ◽  
Type I ◽  
Free System ◽  
Ribosome Inactivating Proteins ◽  
E Coli ◽  
Genes Encoding ◽  
A Cell ◽  
A Subunit

Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in numerous plants, but also constitute a subunit of the bacterial Shiga toxin. Classification of plant RIPs is based on the absence (type I) or presence (type II) of an additional lectin module. In Shiga toxin, sugar binding is mediated by a distinct RIP-associated homopentamer. In the genome of two actinomycetes, we identified RIP-like proteins that resemble plant type I RIPs rather than the RIP subunit (StxA) of Shiga toxin. Some representatives of β- and γ-proteobacteria also contain genes encoding RIP-like proteins, but these are homologous to StxA. Here, we describe the isolation and initial characterization of the RIP-like gene product SCO7092 (RIPsc) from the Gram-positive soil bacterium Streptomyces coelicolor. The ripsc gene was expressed in Escherichia coli as a recombinant protein of about 30 kDa, and displayed the characteristic N-glycosidase activity causing specific rRNA depurination. In Streptomyces lividans and E. coli, RIPsc overproduction resulted in a dramatic decrease in the growth rate. In addition, intracellular production was deleterious for Saccharomyces cerevisiae. However, when applied externally to microbial cells, purified RIPsc did not display antibacterial or antifungal activity, suggesting that it cannot enter these cells. In a cell-free system, however, purified S. coelicolor RIPsc protein displayed strong inhibitory activity towards protein translation.

Preliminary study on preparation of E. coli cell-free system for protein expression

2008 ◽  
Vol 2 (2) ◽  
pp. 224-229 ◽  
Author(s):  
Haiqin Chen ◽  
Zhinan Xu ◽  
Cheng Wang ◽  
Yuhua Liao ◽  
Peilin Cen
Keyword(s):  
Protein Expression ◽  
Coli Cell ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
Preliminary Study

scholarly journals Dynamic Sequence Specific Constraint-Based Modeling of Cell-Free Protein Synthesis

Processes ◽  
2018 ◽  
Vol 6 (8) ◽  
pp. 132 ◽  
Author(s):  
David Dai ◽  
Nicholas Horvath ◽  
Jeffrey Varner
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Protein Production ◽  
Pentose Phosphate ◽  
Free Protein ◽  
E Coli ◽  
Metabolic Fluxes ◽  
Dynamic Constraint ◽  
Flux Estimation ◽  
Constraint Based Modeling

Cell-free protein expression has emerged as an important approach in systems and synthetic biology, and a promising technology for personalized point of care medicine. Cell-free systems derived from crude whole cell extracts have shown remarkable utility as a protein synthesis technology. However, if cell-free platforms for on-demand biomanufacturing are to become a reality, the performance limits of these systems must be defined and optimized. Toward this goal, we modeled E. coli cell-free protein expression using a sequence specific dynamic constraint-based approach in which metabolite measurements were directly incorporated into the flux estimation problem. A cell-free metabolic network was constructed by removing growth associated reactions from the iAF1260 reconstruction of K-12 MG1655 E. coli. Sequence specific descriptions of transcription and translation processes were then added to this metabolic network to describe protein production. A linear programming problem was then solved over short time intervals to estimate metabolic fluxes through the augmented cell-free network, subject to material balances, time rate of change and metabolite measurement constraints. The approach captured the biphasic cell-free production of a model protein, chloramphenicol acetyltransferase. Flux variability analysis suggested that cell-free metabolism was potentially robust; for example, the rate of protein production could be met by flux through the glycolytic, pentose phosphate, or the Entner-Doudoroff pathways. Variation of the metabolite constraints revealed central carbon metabolites, specifically upper glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate, to be the most effective at training a predictive model, while energy and amino acid measurements were less effective. Irrespective of the measurement set, the metabolic fluxes (for the most part) remained unidentifiable. These findings suggested dynamic constraint-based modeling could aid in the design of cell-free protein expression experiments for metabolite prediction, but the flux estimation problem remains challenging. Furthermore, while we modeled the cell-free production of only a single protein in this study, the sequence specific dynamic constraint-based modeling approach presented here could be extended to multi-protein synthetic circuits, RNA circuits or even small molecule production.

scholarly journals Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator

2019 ◽  
Vol 2 (2) ◽  
pp. 52 ◽  
Author(s):  
Seung-Ook Yang ◽  
Gregory H. Nielsen ◽  
Kristen M. Wilding ◽  
Merideth A. Cooper ◽  
David W. Wood ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Wholesale Price ◽  
Free System ◽  
Free Protein ◽  
E Coli ◽  
On Demand ◽  
Tissue Plasminogen ◽  
Cell Free Protein Synthesis

Stroke is the leading cause of death with over 5 million deaths worldwide each year. About 80% of strokes are ischemic strokes caused by blood clots. Tissue plasminogen activator (tPa) is the only FDA-approved drug to treat ischemic stroke with a wholesale price over $6000. tPa is now off patent although no biosimilar has been developed. The production of tPa is complicated by the 17 disulfide bonds that exist in correctly folded tPA. Here, we present an Escherichia coli-based cell-free protein synthesis platform for tPa expression and report conditions which resulted in the production of active tPa. While the activity is below that of commercially available tPa, this work demonstrates the potential of cell-free expression systems toward the production of future biosimilars. The E. coli-based cell-free system is increasingly becoming an attractive platform for low-cost biosimilar production due to recent developments which enable production from shelf-stable lyophilized reagents, the removal of endotoxins from the reagents to prevent the risk of endotoxic shock, and rapid on-demand production in hours.

scholarly journals Akaby - cell-free protein expression system for linear templates

2021 ◽  
Author(s):  
Wakana Sato ◽  
Judee Sharon ◽  
Christopher Deich ◽  
Nathaniel Gaut ◽  
Brock Cash ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression System ◽  
Nuclease Activity ◽  
Coli Strain ◽  
Free Expression ◽  
Dna Templates ◽  
Free Protein ◽  
E Coli ◽  
Linear Dna ◽  
Simple Alternative

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.

scholarly journals Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Green Fluorescence Protein ◽  
T7 Rna Polymerase ◽  
Cell Factory ◽  
T7 Promoter ◽  
Iptg Induction ◽  
E Coli ◽  
A Cell

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy. Results Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively. Conclusion Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.

scholarly journals Sequence Specific Modeling ofE. coliCell-Free Protein Synthesis

2017 ◽  
Author(s):  
Michael Vilkhovoy ◽  
Nicholas Horvath ◽  
Che-Hsiao Shih ◽  
Joseph A. Wayman ◽  
Kara Calhoun ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Training Data ◽  
Model Parameters ◽  
Free System ◽  
Free Protein ◽  
E Coli ◽  
Constraint Based Modeling ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we used sequence specific constraint based modeling to evaluate the performance ofE. colicell-free protein synthesis. A coreE. colimetabolic network, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid biosynthesis and degradation was augmented with sequence specific descriptions of transcription and translation and effective models of promoter function. Model parameters were largely taken from literature, thus the constraint based approach coupled the transcription and translation of the protein product, and the regulation of gene expression, with the availability of metabolic resources using only a limited number of adjustable model parameters. We tested this approach by simulating the expression of two model proteins: chloramphenicol acetyltransferase and dual emission green fluorescent protein, for which we have training data sets; we then expanded the simulations to a range of additional proteins. Protein expression simulations were consistent with measurements for a variety of cases. The constraint based simulations confirmed that oxidative phosphorylation was active in the CAT cell-free extract, as without it there was no feasible solution within the experimental constraints of the system. We then compared the metabolism of theoretically optimal and experimentally constrained CFPS reactions, and developed parameter free correlations which could be used to estimate productivity as a function of protein length and promoter type. Lastly, global sensitivity analysis identified the key metabolic processes that controlled CFPS productivity and energy efficiency. In summary, sequence specific constraint based modeling of CFPS offered a novel means toa prioriestimate the performance of a cell-free system, using only a limited number of adjustable parameters. While we modeled the production of a single protein in this study, the approach could easily be extended to multi-protein synthetic circuits, RNA circuits or the cell free production of small molecule products.

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