Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

Mital, Suraj; Christie, Graham; Dikicioglu, Duygu

doi:10.1186/s12934-021-01698-w

Cited by 31 publications

(22 citation statements)

References 191 publications

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“…The pET28a-PMMoV CP-His vector was obtained from GenScript Biotech Corporation (Nanjing, China). The vector contains the PMMoV CP gene (474 bp) with a histidine tag in its c-terminus, a kanamycin resistance gene, and a T7 promoter . Transformation was verified by colony PCR and agarose gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Pepper Mild Mottle Virus Coat Protein as a Novel Target to Screen Antiviral Drugs

Shi

Liu

et al. 2022

J. Agric. Food Chem.

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Pepper mild mottle virus (PMMoV) has caused serious economic losses to crop production in many countries. The coat protein (CP) of PMMoV is a multifunctional protein proved to be a determining factor in the assignment of virulence type. Therefore, we studied the interaction between drugs and PMMoV CP as a method to screen anti-PMMoV agents. In this study, vanisulfane (6f) exhibited good inactivation activity (68.5%) by biological activity screening. Meanwhile, the green fluorescent protein and PMMoV CP expression changes of vanisulfane against PMMoV were verified by western blot and qRT-PCR experiments. The affinity between vanisulfane and PMMoV CP was predicted to be the best by autodocking and molecular dynamics simulation. PMMoV CP was purified for the first time from the soluble fraction, and the strong affinity between vanisulfane and CP was further verified by interaction experiments. Therefore, this study found that vanisulfane is a potential anti-PMMoV drug targeting PMMoV CP.

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Section: Methodsmentioning

confidence: 99%

Pepper Mild Mottle Virus Coat Protein as a Novel Target to Screen Antiviral Drugs

Shi

Liu

et al. 2022

J. Agric. Food Chem.

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“…Although the use of large insert metagenomic libraries has the potential to counteract some of these challenges, problems with heterologous gene expression in the surrogate host, e.g., failed gene expression and incorrect post-transcriptional processing, remain one of the major limitations of functional metagenomic approaches ( Perner et al, 2011b ; Johnson et al, 2017 ). The use of custom expression strains, alternative vector systems, ionic liquids, or even in vitro recombinant transcription systems are promising techniques to mitigate these shortcomings ( Lam et al, 2015 ; Kinfu et al, 2017 ; Mital et al, 2021 ).…”

Section: Metagenomics: Towards Understanding the Metabolic Microbial ...mentioning

confidence: 99%

Approaches to Unmask Functioning of the Uncultured Microbial Majority From Extreme Habitats on the Seafloor

Böhnke

Perner

2022

Front. Microbiol.

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Researchers have recognized the potential of enzymes and metabolic pathways hidden among the unseen majority of Earth’s microorganisms for decades now. Most of the microbes expected to colonize the seafloor and its subsurface are currently uncultured. Thus, their ability and contribution to element cycling remain enigmatic. Given that the seafloor covers ∼70% of our planet, this amounts to an uncalled potential of unrecognized metabolic properties and interconnections catalyzed by this microbial dark matter. Consequently, a tremendous black box awaits discovery of novel enzymes, catalytic abilities, and metabolic properties in one of the largest habitats on Earth. This mini review summarizes the current knowledge of cultivation-dependent and -independent techniques applied to seafloor habitats to unravel the role of the microbial dark matter. It highlights the great potential that combining microbiological and biogeochemical data from in situ experiments with molecular tools has for providing a holistic understanding of bio-geo-coupling in seafloor habitats and uses hydrothermal vent systems as a case example.

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“…Since the recombinant chitosanase levels were affected by induction conditions, the effects of different expression temperatures, inducer concentrations, and induction time on the production of CHOE were determined in the study [27]. The CHOE production by the recombinant strains of E. coli BL (21) DE3/pET 28a(+)-choe were detected under the induction conditions, IPTG concentrations of 0.025-0.70 mM and temperature range of 16-37 °C.…”

Section: Expressing Of Choe and Production Of Choementioning

confidence: 99%

Gene cloning and molecular characterization of a thermostable chitosanase from Bacillus cereus TY24

Zhang

et al. 2022

BMC Biotechnol

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Background An important conceptual advance in health and the environment has been recognized that enzymes play a key role in the green processing industries. Of particular interest, chitosanase is beneficial for recycling the chitosan resource and producing chitosan oligosaccharides. Also, chitosan gene expression and molecular characterization will promote understanding of the biological function of bacterial chitosanase as well as explore chitosanase for utilizing chitosan resources. Results A chitosanase-producing bacterium TY24 was isolated and identified as Bacillus cereus. Moreover, the chitosanase gene was cloned and expressed in Escherichia coli. Sequence analysis reveals that the recombinant chitosanase (CHOE) belongs to the glycoside hydrolases 8 family. The purified CHOE has a molecular weight of about 48 kDa and the specific activity of 1150 U/mg. The optimal pH and temperature of CHOE were 5.5 and 65 °C, respectively. The enzyme was observed stable at the pH range of 4.5–7.5 and the temperature range of 30–65 °C. Especially, the half-life of CHOE at 65 °C was 161 min. Additionally, the activity of CHOE was remarkably enhanced in the presence of Mn2+, Cu2+, Mg2+ and K+, beside Ca2+ at 5 mM. Especially, the activity of CHOE was enhanced to more than 120% in the presence of 1% of various surfactants. CHOE exhibited the highest substrate specificity toward colloid chitosan. Conclusion A bacterial chitosanase was cloned from B. cereus and successfully expressed in E. coli (BL21) DE3. The recombinant enzyme displayed good stability under acid pH and high-temperature conditions.

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Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

Cited by 31 publications

References 191 publications

Pepper Mild Mottle Virus Coat Protein as a Novel Target to Screen Antiviral Drugs

Pepper Mild Mottle Virus Coat Protein as a Novel Target to Screen Antiviral Drugs

Approaches to Unmask Functioning of the Uncultured Microbial Majority From Extreme Habitats on the Seafloor

Gene cloning and molecular characterization of a thermostable chitosanase from Bacillus cereus TY24

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