scholarly journals Cascaded processing enables continuous upstream processing with E. coli BL21(DE3)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Stefan Kittler ◽  
Christoph Slouka ◽  
Andreas Pell ◽  
Roman Lamplot ◽  
Mihail Besleaga ◽  
...  
Keyword(s):  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Continuous Cultivation ◽  
Continuous Processes ◽  
E Coli ◽  
Industrial Sectors ◽  
Golden Standard ◽  
Cultivation Strategy ◽  
Upstream Processing

AbstractIn many industrial sectors continuous processing is already the golden standard to maximize productivity. However, when working with living cells, subpopulation formation causes instabilities in long-term cultivations. In cascaded continuous cultivation, biomass formation and recombinant protein expression can be spatially separated. This cultivation mode was found to facilitate stable protein expression using microbial hosts, however mechanistic knowledge of this cultivation strategy is scarce. In this contribution we present a method workflow to reduce workload and accelerate the establishment of stable continuous processes with E. coli BL21(DE3) exclusively based on bioengineering methods.

scholarly journals pTSara-NatB, an improved N-terminal acetylation system for recombinant protein expression in E. coli

PLoS ONE ◽  
2018 ◽  
Vol 13 (7) ◽  
pp. e0198715 ◽  
Author(s):  
Matteo Rovere ◽  
Alex Edward Powers ◽  
Dushyant Shailesh Patel ◽  
Tim Bartels
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
E Coli

Effect of supplementing Moringa oleifera essential oils on milk quality and fatty acid profile in dairy sheep

2019 ◽  
Author(s):  
N. Hemamalini ◽  
S. Ezhilmathi ◽  
A. Angela Mercy
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Recombinant Protein Expression ◽  
Coli Strain ◽  
Expression Vectors ◽  
Functional Domain ◽  
Post Translational Modifications ◽  
E Coli

Escherichia coli is the most extensively used organism in recombinant protein production. It has several advantages including a very short life cycle, ease of genetic manipulation and the well-known cell biology etc. which makes E. coli as the perfect host for recombinant protein expression. Despite many advantages, E. coli also have few disadvantages such as coupled transcription and translation and lack of eukaryotic post-translational modifications. These challenges can be overcome by adopting several strategies such as, using different E. coli expression vectors, changing the gene sequence without altering the functional domain, modified E. coli strain usage, changing the culture parameters and co-expression with a molecular chaperone. In this review, we present the level of strategies used to enhance the recombinant protein expression and its stability in E. coli.

Monitoring of transcriptome and proteome profiles to investigate the cellular response of E. coli towards recombinant protein expression under defined chemostat conditions

2008 ◽  
Vol 135 (1) ◽  
pp. 34-44 ◽  
Author(s):  
Karin Dürrschmid ◽  
Helga Reischer ◽  
Wolfgang Schmidt-Heck ◽  
Thomas Hrebicek ◽  
Reinhard Guthke ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Cellular Response ◽  
Recombinant Protein Expression ◽  
E Coli

scholarly journals Codon Preference Optimization Increases Prokaryotic Cystatin C Expression

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Qing Wang ◽  
Cui Mei ◽  
Honghua Zhen ◽  
Jess Zhu
Keyword(s):  
Protein Expression ◽  
Cystatin C ◽  
Production Systems ◽  
Codon Optimization ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Gc Content ◽  
Optimization Techniques ◽  
E Coli ◽  
Prokaryotic Expression Vector

Gene expression is closely related to optimal vector-host system pairing in many prokaryotes. Redesign of the humancystatin C(cysC) gene using the preferred codons of the prokaryotic system may significantly increasecysCexpression inEscherichia coli(E. coli). Specifically,cysCexpression may be increased by removing unstable sequences and optimizing GC content. According toE. coliexpression system codon preferences, the gene sequence was optimized while the amino acid sequence was maintained. The codon-optimizedcysC(co-cysC) and wild-typecysC(wt-cysC) were expressed by cloning the genes into a pET-30a plasmid, thus transforming the recombinant plasmid intoE. coliBL21. Before and after the optimization process, the prokaryotic expression vector and host bacteria were examined for protein expression and biological activation of CysC. The recombinant proteins in the lysate of the transformed bacteria were purified using Ni2+-NTA resin. Recombinant protein expression increased from 10% to 46% based on total protein expression after codon optimization. Recombinant CysC purity was above 95%. The significant increase incysCexpression inE. coliexpression produced by codon optimization techniques may be applicable to commercial production systems.

scholarly journals Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

2021 ◽  
Vol 12 ◽  
Author(s):  
Gema Lozano Terol ◽  
Julia Gallego-Jara ◽  
Rosa Alba Sola Martínez ◽  
Adrián Martínez Vivancos ◽  
Manuel Cánovas Díaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Copy Number ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

scholarly journals Escherichia coli robustly expresses ATP synthase at growth rate maximizing concentrations

2021 ◽  
Author(s):  
Iraes Rabbers ◽  
Frank J Bruggeman
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Protein Expression ◽  
Genetic Variants ◽  
Evolutionary Adaptation ◽  
Wild Type ◽  
Short Term ◽  
E Coli ◽  
Fitness Maximization

AbstractImproved protein expression is an important evolutionary adaptation of bacteria. A key question is whether evolution has led to optimal protein expression that maximizes immediate growth rate (short-term fitness) across conditions. Alternatively, fitter genetic variants could display suboptimal short-term fitness, because they cannot do better or because they strive for long-term fitness maximization by, for instance, anticipating future conditions. To answer this question, we focus on the ATP-producing enzyme F1F0 H+-ATPase, which is an abundant enzyme and ubiquitously expressed across conditions. We tested the optimality of H+-ATPase expression in Escherichia coli across 27 different nutrient conditions. In all tested conditions, wild-type E. coli expresses its H+- ATPase remarkably close to optimal concentrations that maximize immediate growth rate. This work indicates that bacteria can achieve robust optimal protein expression for immediate growth- rate.

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