Biotechnological applications of periplasmic expression in E. coli

2012 ◽  
Vol 01 (02) ◽  
Author(s):  
Edwin van Bloois
Keyword(s):  
Biotechnological Applications ◽  
E Coli ◽  
Periplasmic Expression

Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

2019 ◽  
Vol 20 (6) ◽  
pp. 497-505 ◽  
Author(s):  
Abeer M. Abd El-Aziz ◽  
Mohamed A. Shaker ◽  
Mona I. Shaaban
Keyword(s):  
Gold Nanoparticles ◽  
Lipolytic Activity ◽  
Market Demand ◽  
Biotechnological Applications ◽  
Lipase Gene ◽  
Expression Plasmid ◽  
E Coli ◽  
Nickel Affinity Chromatography ◽  
Recombinant Production ◽  
Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
Author(s):  
Jörg F. Rippmann ◽  
Michaela Klein ◽  
Christian Hoischen ◽  
Bodo Brocks ◽  
Wolfgang J. Rettig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Binding Activity ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Active Product ◽  
Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

Effect of DnaK/DnaJ/GrpE and DsbC Chaperons on Periplasmic Expression of Fab Antibody by E. coli SEC Pathway

2017 ◽  
Vol 25 (1) ◽  
pp. 67-74 ◽  
Author(s):  
Hassan Dariushnejad ◽  
Safar Farajnia ◽  
Nosratollah Zarghami ◽  
Maryam Aria ◽  
Asghar Tanomand
Keyword(s):  
E Coli ◽  
Periplasmic Expression ◽  
Sec Pathway

The journey of Deinococcus radiodurans; a perspective

2021 ◽  
Vol 11 ◽  
Author(s):  
Gajendra Mohan Baldodiya
Keyword(s):  
Deinococcus Radiodurans ◽  
Model Organism ◽  
Genotoxic Stress ◽  
Severe Drought ◽  
Radiation Tolerance ◽  
X Rays ◽  
Biotechnological Applications ◽  
E Coli ◽  
World Records ◽  
Post Stress

: Deinococcus radiodurans has been recognized for its robustness and recorded in the Guinness Book of World Records as the world's toughest known bacterium. In essence, the title comes from its ability to survive extreme conditions such as severe drought (desiccation) and radiation tolerance up to 15000 Gy, which is more than 250 times of E. coli and about 3000 times of humans. Due to its high tolerance to all kinds of genotoxic stress, such as desiccation, UV, X-rays, and oxidants, D. radiodurans is a well-suited model organism for microbial radiation resistance studies. The DNA damage-responsive gene expression is an important component of post-stress recovery where the cell shows a great multiplicity of genomes leading to the highly proficient recombinational DNA repair. This article pitches light on the unique properties of D. radiodurans, unfolding its journey so far as well as important molecular discoveries, prospects, and biotechnological applications.

scholarly journals Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation – Suggestion of a Binding Mode at the α7 nAChR

2019 ◽  
Vol 10 ◽  
Author(s):  
Yamina El Hamdaoui ◽  
Xiaosa Wu ◽  
Richard J. Clark ◽  
Julien Giribaldi ◽  
Raveendra Anangi ◽  
...  
Keyword(s):  
Binding Mode ◽  
E Coli ◽  
Periplasmic Expression ◽  
Α7 Nachr

scholarly journals Sponge-Like: A New Protocol for Preparing Bacterial Ghosts

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Amro A. Amara ◽  
Mounir M. Salem-Bekhit ◽  
Fars K. Alanazi
Keyword(s):  
Gel Electrophoresis ◽  
Chemical Compounds ◽  
Agarose Gel Electrophoresis ◽  
Biotechnological Applications ◽  
E Coli ◽  
Viable Cells ◽  
Inhibition Concentration ◽  
Active Chemical ◽  
Bacterial Ghosts ◽  
First Time

Bacterial Ghosts (BGs) received an increasing interest in the recent years for their promising medicinal and pharmaceutical applications. In this study, for the first time we introduce a new protocol for BGs production.E. coliBL21 (DE3) pLysS (Promega) was used as a model to establish a general protocol for BGs preparation. The protocol is based on using active chemical compounds in concentrations less than the Minimum Inhibition Concentration (MIC). Those chemical compounds are SDS, NaOH, and H2O2. Plackett-Burman experimental design was used to map the best conditions for BGs production. Normal and electronic microscopes were used to evaluate the BGs quality (BGQ). Spectrophotometer was used to evaluate the amount of the released protein and DNA. Agarose gel electrophoresis was used to determine the existence of any residue of DNA after each BGs preparation. Viable cells, which existed after running this protocol, were subjected to lysis by inducing the lysozyme gene carried on pLysS plasmid. This protocol is able to produce BGs that can be used in different biotechnological applications.

scholarly journals From Data Mining of Chitinophaga sp. Genome to Enzyme Discovery of a Hyperthermophilic Metallocarboxypeptidase

2021 ◽  
Vol 9 (2) ◽  
pp. 393
Author(s):  
Gabriela Cabral Fernandes ◽  
Elwi Guillermo Machado Sierra ◽  
Paul Brear ◽  
Mariana Rangel Pereira ◽  
Eliana G. M. Lemos
Keyword(s):  
Data Mining ◽  
Bacterial Consortium ◽  
Industrial Applications ◽  
Biotechnological Applications ◽  
Negative Bacterium ◽  
Conserved Motif ◽  
E Coli ◽  
Enzyme Discovery ◽  
Ethanol Plant ◽  
Positive Effect

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.

Identification of Translationally Optimal Codons and Suitable Expression Host of DPV gB Gene

2011 ◽  
Vol 343-344 ◽  
pp. 729-736
Author(s):  
Long Jiang ◽  
An Chun Cheng ◽  
Ming Shu Wang ◽  
De Kang Zhu ◽  
Ren Yong Jia
Keyword(s):  
Amino Acids ◽  
Codon Usage ◽  
Biotechnological Applications ◽  
E Coli ◽  
Optimal Codons ◽  
Codon Composition ◽  
Expression Host ◽  
Species Evolution ◽  
High Level ◽  
Suitable Expression

In this report, we conduct study on codon composition and codon usage of DPV glycoprotein B (gB) gene, its homologs constitute the most highly conserved family of herpesvirus glycoproteins and are present in members of each herpesvirus subfamily. Our results show that sixty-one codons (excluding the termination codons) in the polypeptide, a high level of diversity in codon usage bias existed for coding the Ala, Gly, Leu, Pro, Arg, Ser, Thr and Val amino acids. Sixteen codons (each for a amino acid), including GCA (Ala), GAT (Asp), GAA (Glu), GGA (Gly), CAT (His), ATA (Ile), AAA (Lys), CTA (Leu), AAT (Asn), CCA (Pro), CAA (Gln), AGA (Arg), TCT (Ser), ACT (Thr), GTA (Val) and TAT (Tyr) were determined as the translationally optimal codons. The codon preferences of DPV gB gene were compared with those of E. coli, yeast, and H. sapiens, we can speculate that the DPV gB gene may be more efficiently expressed in the E. coli system. In summary, knowledge of codon usage of herpesvirus gB genes provides insights into molecular and species evolution, and also plays important role in furthering some biotechnological applications. These would be fruitful areas for further study.

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