scholarly journals Optimization of Conditions for Maximal Production of Recombinant Thermostable Cellulase from Thermotoga naphthophila using E. coli BL21-CodonPlus (DE3) as Expression Host

2019 ◽  
Vol 51 (4) ◽  
Author(s):  
Aisha Khalid ◽  
Muhammad Tayyab ◽  
Abu Saeed Hashmi ◽  
Tahir Yaqub ◽  
Ali Raza Awan ◽  
...  
Keyword(s):  
E Coli ◽  
Expression Host ◽  
Thermostable Cellulase ◽  
Maximal Production

Peptide nucleic acid restores colistin susceptibility through modulation of MCR-1 expression in Escherichia coli

2020 ◽  
Author(s):  
Xiaoming Wang ◽  
Yao Wang ◽  
Zhuoren Ling ◽  
Chaoyang Zhang ◽  
Mingming Fu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Peptide Nucleic Acid ◽  
Promoter Activity ◽  
Sequence Variation ◽  
Promoter Sequence ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Expression Host ◽  
Antisense Approach ◽  
Increased Susceptibility

Abstract Background Plasmid-mediated mechanisms of drug resistance accelerate the spread of polymyxin resistance, leaving clinicians with few or no antibacterial options for the treatment of infections caused by MDR bacteria, especially carbapenemase-producing strains. Objectives To evaluate the associations among promoter sequence variation, mcr-1 expression, host factors and levels of colistin resistance and to propose antisense agents such as peptide nucleic acids (PNAs) targeting mcr-1 as a tool to restore colistin susceptibility through modulation of MCR-1 expression in Escherichia coli. Methods A β-galactosidase assay was performed to study mcr-1 promoter activity. Quantitative real-time PCR and western blot assays were used to identify the expression level of MCR-1 in WT strains and transformants. Three PNAs targeting different regions of mcr-1 were designed and synthesized to determine whether they can effectively inhibit MCR-1 expression. MIC was measured to test colistin susceptibility in the presence or absence of PNA-1 in mcr-1-carrying E. coli. Results Variation in the mcr-1 promoter sequence and host species affect promoter activity, MCR-1 expression levels and colistin MICs. One PNA targeting the ribosome-binding site fully inhibited the expression of mcr-1 at a concentration of 4 μM, resulting in significantly increased susceptibility to colistin. The MIC90 of colistin decreased from 8 to 2 mg/L (P < 0.05) in the presence of 4 μM PNA. Conclusions These findings suggest that the antisense approach is a possible strategy to combat mcr-1-mediated resistance as well as other causes of emerging global resistance.

Heat treatment purification of thermostable cellulase and hemicellulase enzymes expressed in E. coli

1989 ◽  
Vol 11 (2) ◽  
pp. 113-115 ◽  
Author(s):  
M.L. Patchett ◽  
T.L. Neal ◽  
L.R. Schofield ◽  
R.C. Strange ◽  
R.M. Daniel ◽  
...  
Keyword(s):  
Heat Treatment ◽  
E Coli ◽  
Thermostable Cellulase

scholarly journals A programmable CRISPR/Cas9-based phage defense system for Escherichia coli BL21(DE3)

2020 ◽  
Author(s):  
Li Liu ◽  
Dongdong Zhao ◽  
Lijun Ye ◽  
Tao Zhan ◽  
Bin Xiong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Critical Role ◽  
Viable Cell ◽  
Control Culture ◽  
Phage Infection ◽  
Defense System ◽  
E Coli ◽  
Expression Host ◽  
Order Of Magnitude ◽  
T7 Phage

Abstract Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that it increased the ability of BL21 to defend against phage infection. Furthermore, the targeted loci played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 hours of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% the rate of the un-infected control, which was significantly higher than the parent BL21. We successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21’s to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection.

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 Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor

2017 ◽  
Vol 15 (1) ◽  
pp. 179-185 ◽  
Author(s):  
Subramani Ramkumar ◽  
Vaishnavo Rabindranath Pai ◽  
Chinnathambi Thangadurai ◽  
Vidhya Priya Murugan
Keyword(s):  
Tumor Necrosis Factor ◽  
Comparative Study ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Receptor ◽  
E Coli ◽  
Chemical Complexity ◽  
Expression Host ◽  
Necrosis Factor

scholarly journals The Effect of Single Co-expression of The DnaK-DnaJ-GrpE and GroEL/ES Chaperones and Their Combination on Expression Intein-pretrombin-2 in Escherichia coli ER2566

2020 ◽  
Vol 6 (1) ◽  
pp. 47-54
Author(s):  
Iman Permana Maksum ◽  
D Agus Yusuf Wildan ◽  
Khomaini Hasan ◽  
Toto Subroto
Keyword(s):  
Inclusion Bodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Iptg Induction ◽  
E Coli ◽  
Expression Host ◽  
Sds Page ◽  
Culture Growth ◽  
The One ◽  
Cell Culture Growth

The use of recombinant thrombin in the manufacture of fibrin glue allows diseases contamination to be avoided. However, the expression of recombinant protein in E. coli still has a disadvantage of the formation of inclusion bodies, so it needs to be minimized by co-expression of chaperones. Therefore, the aim of this study was to determine the effect of single DnaK-DnaJ-GrpE and GroEL/ES chaperone expression and their combination on the expression of intein-pretrombin-2Ti,pH on E. coli ER2566. The method started with isolation of pTWIN1-prethrombin-2Ti,pH and pG-KJE8 from E. coli TOP10F' and DH5α respectively, the co-transformation of the expression host E. coli ER2566 using pG-KJE8 and pTWIN1-prethrombin-2Ti,pHvectors, the chaperone co-expression was induced using L-Arabinosa before IPTG induction and cell culture growth was incubated at 22 oC. The expression products were characterized by using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results of the co-expression of chaperone showed that the number of soluble fraction was higher than the one without co-expression of chaperone. In addition, the co-expression of chaperone using pG-KJE8 in intein-prothrombin-2Ti,pH expression was sufficient using tetracycline as an inducer.

scholarly journals Expression and production optimization of the cationic antimicrobial peptide - indolicidin by the recombinant E. coli C41 (DE3) clones

2018 ◽  
Vol 62 (1) ◽  
pp. 61-66
Author(s):  
Kanesan Panneer Selvam ◽  
Guruvu Nambirajan ◽  
Balasubramaniam Annamalai ◽  
Mohammed Alaidarous ◽  
Bader Mohammed Alshehri ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Production Optimization ◽  
Microbial Pathogens ◽  
Cationic Antimicrobial Peptide ◽  
E Coli ◽  
Inhibitory Activities ◽  
Bovine Neutrophils ◽  
Maximal Production ◽  
Level Production ◽  
Broad Type

The cytoplasmic granules of bovine neutrophils naturally possess indolicidin - a promising cationic antimicrobial peptide as it displays inherent inhibitory activities against a broad type of microbial pathogens. In this study, a shake flask level production and expression optimizations of the indolicidin by the recombinant Escherichia coli C41 (DE3) clones (transformed with pET21a(+) plasmid carrying indolicidin gene) were carried out under standard conditions, as to determine the conditions required for maximal production. It was determined that a concentration of 1 mM of IPTG was effective, the 2×YT with salts and LB media at pH 7.5 with 3-6 h of incubation were required for maximal indolicidin expression.

scholarly journals Vibrio natriegens as a pET-Compatible Expression Host Complementary to Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiaqi Xu ◽  
Feng Dong ◽  
Meixian Wu ◽  
Rongsheng Tao ◽  
Junjie Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Catalytic Efficiency ◽  
Consensus Protocol ◽  
E Coli ◽  
Expression Host ◽  
Terrific Broth Medium ◽  
Living Bacterium ◽  
Free Living Bacterium

Efficient and novel recombinant protein expression systems can further reduce the production cost of enzymes. Vibrio natriegens is the fastest growing free-living bacterium with a doubling time of less than 10 min, which makes it highly attractive as a protein expression host. Here, 196 pET plasmids with different genes of interest (GOIs) were electroporated into the V. natriegens strain VnDX, which carries an integrated T7 RNA polymerase expression cassette. As a result, 65 and 75% of the tested GOIs obtained soluble expression in V. natriegens and Escherichia coli, respectively, 20 GOIs of which showed better expression in the former. Furthermore, we have adapted a consensus “what to try first” protocol for V. natriegens based on Terrific Broth medium. Six sampled GOIs encoding biocatalysts enzymes thus achieved 50–128% higher catalytic efficiency under the optimized expression conditions. Our study demonstrated V. natriegens as a pET-compatible expression host with a spectrum of highly expressed GOIs distinct from E. coli and an easy-to-use consensus protocol, solving the problem that some GOIs cannot be expressed well in E. coli.

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