scholarly journals In silico Design of Linear DNA for Robust Cell-Free Gene Expression

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinjie Chen ◽  
Yuan Lu
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
High Throughput Screening ◽  
Expression System ◽  
Dna Templates ◽  
Linear Dna ◽  
Low Protein ◽  
Cell Extracts ◽  
Free Gene ◽  
Expression Templates

Cell-free gene expression systems with linear DNA expression templates (LDETs) have been widely applied in artificial cells, biochips, and high-throughput screening. However, due to the degradation caused by native nucleases in cell extracts, the transcription with linear DNA templates is weak, thereby resulting in low protein expression level, which greatly limits the development of cell-free systems using linear DNA templates. In this study, the protective sequences for stabilizing linear DNA and the transcribed mRNAs were rationally designed according to nucleases’ action mechanism, whose effectiveness was evaluated through computer simulation and cell-free gene expression. The cell-free experiment results indicated that, with the combined protection of designed sequence and GamS protein, the protein expression of LDET-based cell-free systems could reach the same level as plasmid-based cell-free systems. This study would potentially promote the development of the LDET-based cell-free gene expression system for broader applications.

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 Enhancement of transient erythropoietin protein expression by valproic acid in CHO‐K1 suspension adapted cells

2020 ◽  
Vol 25 (1) ◽  
pp. 28
Author(s):  
Yana Rubiyana ◽  
Retno Damajanti Soejoedono ◽  
Adi Santoso
Keyword(s):  
Gene Expression ◽  
Valproic Acid ◽  
Protein Expression ◽  
Histone Deacetylase Inhibitors ◽  
Expression System ◽  
Transient Gene Expression ◽  
Optimum Concentration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Stable Gene ◽  
Gene Expression System

Erythropoietin (EPO) is a therapeutic protein that is widely used to increase red blood cell production in chronic kidney failure. EPO protein can be produced quickly with a transient gene expression system (TGE). However, the titer produced using TGE is usually lower than the stable gene expression system (SGE). It has been known that TGE can be improved by histone deacetylase inhibitors (iHDACs) such as valproic acid (VPA). This study was conducted to examine the VPA effect on EPO protein expression in CHO‐K1 suspension adapted cells and to find the optimum concentration of VPA on transient EPO protein production. EPO proteins was quantified using the enzyme‐linked immunosorbent assay (ELISA) method. The optimization of VPA concentrations showed that VPA increased the EPO protein yield by up to 2‐fold in transient EPO production, and the optimum concentration of VPA was 4 mM. VPA optimization was very helpful to obtain the maximum increase in the transiently expressed protein. Furthermore, this study can be used as a model to produce EPO proteins or other recombinant proteins rapidly with TGE of CHO‐K1 suspension adapted cells.

scholarly journals T7Max transcription system

2021 ◽  
Author(s):  
Christopher Deich ◽  
Brock Cash ◽  
Wakana Sato ◽  
Judee Sharon ◽  
Lauren Aufdembrink ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Polymerase ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Dna Templates ◽  
Transcription System ◽  
In Vitro Systems ◽  
In Vitro Protein Expression ◽  
Rna Polymerase Promoter

Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. Here we present a modified T7 RNA polymerase promoter that acts to significantly increase the yields of both transcription and translation within in vitro systems. The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system. Unlike other methods of limiting linear template degradation, the T7Max promoter increases transcript concentration in a T7 transcription reaction, providing more mRNA for translation.

Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System

Nano Letters ◽  
2018 ◽  
Vol 18 (4) ◽  
pp. 2650-2657 ◽  
Author(s):  
Matthaeus Schwarz-Schilling ◽  
Aurore Dupin ◽  
Fabio Chizzolini ◽  
Swati Krishnan ◽  
Sheref S. Mansy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Gene Expression System ◽  
A Cell ◽  
Free Gene

Expression of Foreign Proteins in Escherichia coli

2021 ◽  
Author(s):  
Ali Iftikhar
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Expression System ◽  
Maximum Yield ◽  
Protein Yield ◽  
Foreign Protein ◽  
Low Protein ◽  
Prokaryotic Expression System ◽  
Foreign Proteins

Abstract BackgroundOptimization of conditions for the recombinant production of proteins in a prokaryotic expression system is essential as the recombinant proteins impose a metabolic burden on cell's growth leading to low protein yield and low protein expression resulting from cell death.Main textThe concentration of media components is optimized to accommodate for depleted nutrients due to foreign protein expression. The temperature is optimized to reduce proteolytic degradation and accumulation of protein as inclusion bodies in Escherichia coli. The concentration of inducer and time of induction for high protein yield is also optimized. These optimization conditions depend on the promoter under which the gene of interest is present and the characteristics of the target protein.ConclusionIn the past few years, many optimization conditions for the production of recombinant proteins in Escherichia coli have been studied. These conditions depend mainly upon the promoter used to produce protein and the type of protein produced. Optimizing the expression parameters of protein produced in Escherichia coli ensures maximum yield of the desired protein.

scholarly journals Self-organization controls expression more than abundance of molecular components of transcription and translation in confined cell-free gene expression

2018 ◽  
Author(s):  
P.M. Caveney ◽  
R. Dabbs ◽  
G. Chauhan ◽  
S.E. Norred ◽  
C.P. Collier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Production ◽  
Self Organization ◽  
Large Variability ◽  
Expression Noise ◽  
Expression Efficiency ◽  
Cell Extracts ◽  
Reaction Chambers ◽  
Free Gene ◽  
Molecular Components

AbstractCell-free gene expression using purified components or cell extracts has become an important platform for synthetic biology that is finding a growing numBer of practical applications. Unfortunately, at cell-relevant reactor volumes, cell-free expression suffers from excessive variability (noise) such that protein concentrations may vary by more than an order of magnitude across a population of identically constructed reaction chambers. Consensus opinion holds that variability in expression is due to the stochastic distribution of expression resources (DNA, RNAP, ribosomes, etc.) across the population of reaction chambers. In contrast, here we find that chamber-to-chamber variation in the expression efficiency generates the large variability in protein production. Through analysis and modeling, we show that chambers self-organize into expression centers that control expression efficiency. Chambers that organize into many centers, each having relatively few expression resources, exhibit high expression efficiency. Conversely, chambers that organize into just a few centers where each center has an abundance of resources, exhibit low expression efficiency. A particularly surprising finding is that diluting expression resources reduces the chamber-to-chamber variation in protein production. Chambers with dilute pools of expression resources exhibit higher expression efficiency and lower expression noise than those with more concentrated expression resources. In addition to demonstrating the means to tune expression noise, these results demonstrate that in cell-free systems, self-organization may exert even more influence over expression than the abundance of the molecular components of transcription and translation. These observations in cell-free platform may elucidate how self-organized, membrane-less structures emerge and function in cells.

scholarly journals Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

2020 ◽  
Author(s):  
Bo Zhu ◽  
Rui Gan ◽  
Maria D. Cabezas ◽  
Takaaki Kojima ◽  
Robert Nicol ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Model Organisms ◽  
Single Chain ◽  
Dna Templates ◽  
E Coli ◽  
Free Gene ◽  
Expression Templates ◽  
Dsdna Binding ◽  
Linear Expression ◽  
Cell Free Protein Synthesis

AbstractIn crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over PCR-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a dsDNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based Protection of LET) method effectively increases sfGFP expression levels from LETs in Escherichia coli CFPS reactions by 6-fold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from non-model organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

scholarly journals A novel gene expression system for Ralstonia eutropha based on the T7 promoter

2020 ◽  
Author(s):  
Changhao Bi ◽  
Bin Xiong ◽  
Muzi Hu ◽  
Zhongkang Li ◽  
Li Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Ralstonia Eutropha ◽  
Expression System ◽  
T7 Promoter ◽  
T7 Expression System ◽  
Plasmid Size ◽  
Rna Polymerase Gene ◽  
Essential Sequence ◽  
Electroporation Efficiency

Abstract Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for the metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression. Although current plasmid systems for R. eutropha provide a basic platform for gene expression, the performance of the induction systems is still limited. In addition, the sizes of the cloned genes is limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into genome of R. eutropha, and cloning the T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted, reducing the expression plasmid size to 3392 bp, and the electroporation efficiency was improved 4 times. As a result, the highest expression level of Rfp was enhanced slightly, and the L-arabinose concentration necessary for induction was decreased 20 times. Conclusions: R. eutropha with the T7 expression system provides an efficient platform for protein expression and synthetic biology applications.

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