scholarly journals Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

2019 ◽  
Author(s):  
Romel Menacho-Melgar ◽  
Zhixia Ye ◽  
Eirik A. Moreb ◽  
Tian Yang ◽  
John P. Efromson ◽  
...  
Keyword(s):  
Protein Expression ◽  
Stationary Phase ◽  
Recombinant Protein Expression ◽  
Cellular Protein ◽  
List Type ◽  
Heterologous Proteins ◽  
Fed Batch ◽  
E Coli ◽  
Dry Cell Weight ◽  
Phosphate Depletion

AbstractWe report the improved production of recombinant proteins in E. coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of total cellular protein. Initial use of the method in instrumented fed batch fermentations enables cell densities of ∼30 grams dry cell weight (gCDW) per liter and protein titers up to 8.1+/−0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μL (384 well plates), 100 μL (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach ∼ 5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.HighlightsStationary phase protein expression results in high titers.Autoinduction by phosphate depletion enables protein titers from 2-8 g/L.Autoinduction has been validated from 384 well plates to instrumented bioreactors.

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 Enhanced production of recombinant serratiopeptidase in Escherichia coli and its characterization as a potential biosimilar to native biotherapeutic counterpart

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Vishal Srivastava ◽  
Shivam Mishra ◽  
Tapan K. Chaudhuri
Keyword(s):  
Protein Expression ◽  
Process Parameters ◽  
Recombinant Expression ◽  
Cellular Protein ◽  
Biologically Active ◽  
Fixed Dose ◽  
Antibiofilm Activity ◽  
Protein Accumulation ◽  
E Coli ◽  
Enhanced Production

Abstract Background Serratia marcescens, a Gram-negative nosocomial pathogen secretes a 50 kDa multi-domain zinc metalloprotease called serratiopeptidase. Broad substrate specificity of serratiopeptidase makes it suitable for detergent and food processing industries The protein shows potent anti-inflammatory, anti-edemic, analgesic, antibiofilm activity and sold as an individual or fixed-dose enteric-coated tablets combined with other drugs. Although controversial, serratiopeptidase as drug is used in the treatment of chronic sinusitis, carpal tunnel syndrome, sprains, torn ligaments, and postoperative inflammation. Since the native producer of serratiopeptidase is a pathogenic microorganism, the current production methods need to be replaced by alternative approaches. Heterologous expression of serratiopeptidase in E. coli was tried before but not found suitable due to the limited yield, and other expression related issues due to its inherent proteolytic activity such as cytotoxicity, cell death, no expression, minimal expression, or inactive protein accumulation. Results Recombinant expression of mature form serratiopeptidase in E. coli seems toxic and resulted in the failure of transformation and other expression related issues. Although E. coli C43(DE3) cells, express protein correctly, the yield was compromised severely. Optimization of protein expression process parameters such as nutrient composition, induction point, inducer concentration, post-induction duration, etc., caused significant enhancement in serratiopeptidase production (57.9 ± 0.73% of total cellular protein). Expressed protein formed insoluble, enzymatically inactive inclusion bodies, and gave 40–45 mg/l homogenous (> 98% purity) biologically active and conformationally similar serratiopeptidase to the commercial counterpart upon refolding and purification. Conclusion Expression of mature serratiopeptidase in E. coli C43(DE3) cells eliminated the protein expression associated with toxicity issues. Further optimization of process parameters significantly enhanced the overexpression of protein resulting in the higher yield of pure and functionally active recombinant serratiopeptidase. The biological activity and conformational features of recombinant serratiopeptidase were very similar to the commercially available counterpart suggesting it-a potential biosimilar of therapeutic and industrial relevance.

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

scholarly journals Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

2020 ◽  
Vol 21 (3) ◽  
pp. 990 ◽  
Author(s):  
Kangsan Kim ◽  
Donghui Choe ◽  
Dae-Hee Lee ◽  
Byung-Kwan Cho
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Recombinant Protein Expression ◽  
Cost Effective ◽  
Protein Yield ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Expression Pathways

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

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 Improved, two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

2020 ◽  
Author(s):  
Romel Menacho-Melgar ◽  
Eirik A. Moreb ◽  
John P. Efromson ◽  
Michael D. Lynch
Keyword(s):  
Protein Expression ◽  
Membrane Integrity ◽  
Heterologous Protein Expression ◽  
List Type ◽  
Two Stage ◽  
Microtiter Plates ◽  
Thaw Cycle ◽  
Protein Expression And Purification ◽  
Phosphate Depletion ◽  
Rna Endonuclease

AbstractWe report improved release of recombinant proteins in E. coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the non specific DNA/RNA endonuclease from S. marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. Post cell harvest, the addition of detergent (0.1% Triton-X100) and a single 30 minutes freezer thaw cycle results in > 90% release of protein (GFP). This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high throughput cultivation in microtiter plates and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.HighlightsAutoinduction of both cell lysis and nucleotide hydrolysis>90 % lysis and DNA degradationStrains are stable to lysis in the absence of phosphate depletion.

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
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