scholarly journals Scalable, two‐stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

2020 ◽  
Vol 117 (9) ◽  
pp. 2715-2727 ◽  
Author(s):  
Romel Menacho‐Melgar ◽  
Zhixia Ye ◽  
Eirik A. Moreb ◽  
Tian Yang ◽  
John P. Efromson ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Two Stage ◽  
E Coli ◽  
Phosphate Depletion

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.

scholarly journals Tunable recombinant protein expression in E. coli: enabler for continuous processing?

2016 ◽  
Vol 100 (13) ◽  
pp. 5719-5728 ◽  
Author(s):  
Lukas Marschall ◽  
Patrick Sagmeister ◽  
Christoph Herwig
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Continuous Processing ◽  
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 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 Multimodal Approaches for the Improvement of the Cellular Folding of a Recombinant Iron Regulatory Protein in E. Coli

2021 ◽  
Author(s):  
Gayathri Ravitchandirane ◽  
Sheetal Bandhu ◽  
Tapan K. Chaudhuri
Keyword(s):  
Heat Shock ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Physiological Stress ◽  
Recombinant Protein Expression ◽  
Host Cells ◽  
Growth Media ◽  
E Coli ◽  
Expression Host

Abstract BackgroundDuring the recombinant protein expression, foreign proteins are generated in insoluble and inactive aggregates in E. coli cell factories, which inhibits E. coli from being employed as an expression host despite its numerous advantages and ease of use. The yeast mitochondrial aconitase protein, which has a tendency to aggregate when expressed in E. coli cells in the absence of heterologous chaperones GroEL/ES was utilised as a model to investigate how the modulation of physiological stimuli in the host cell can increase protein solubility. The process variables such as incubation temperature, inducer concentrations, growth media, and the presence of folding modulators such as exogenous molecular chaperones or osmolytes are crucial for the cellular folding and are investigated in the study. The processes the physiological stress such as osmotic and heat shock stimulation in the host cells and thereby their effect on the solubility and activity of recombinant proteins was also analysed.ResultsOf the various methods discussed, the cells subjected to the addition of osmolytes and pre-induction heat shock exhibited significant enhancement in the recombinant aconitase activity. The concomitant GroEL/ES expression further assists the folding of these soluble aggregates and increases the functional protein molecules in the cytoplasm of the recombinant E. coli cells.ConclusionsThe recombinant E. coli cells enduring physiological stress provide a cytosolic environment for the enhancement in the solubility and activity of the recombinant proteins. GroEL/ES-expressing cells not only aided in the folding of recombinant proteins, but also had an effect on the physiology of the expression host. The improvement in the specific growth rate and aconitase productivity during chaperone GroEL/ES co-expression is attributed to the reduction in overall cellular stress caused by the expression host's aggregation-prone recombinant protein expression.

scholarly journals Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Richa Guleria ◽  
Priyanka Jain ◽  
Madhulika Verma ◽  
Krishna J. Mukherjee
Keyword(s):  
Protein Synthesis ◽  
Stress Response ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Double Knockout ◽  
Down Regulation ◽  
E Coli

Abstract Background A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase. Results We chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. Conclusion The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

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