The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity

Birchfield, Aaron; McIntosh, Cecilia A.

doi:10.3390/plants9030402

Cited by 4 publications

(13 citation statements)

References 51 publications

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Section: Cloning Verification and Transformation Of Cp3gt Into Pichia...mentioning

confidence: 99%

“…A pPicZα plasmid https://www.snapgene.com/plasmids/yeast_plasmids/pPICZ_A (accessed on 10 November 2023) containing the Cp3GT coding region (GQ141630) was transformed into Escherichia coli cells for propagation and grown as previously described [49]. After cultivation, plasmid DNA was isolated and sequenced to confirm the wild-type Cp3GT gene's presence, as previously demonstrated [49]. Using site-directed mutagenesis, a thrombin cleavage sequence (LVPRGS) was integrated upstream of the C-terminal c-myc/6x His recombinant tags, to allow post-purification tag removal (Supplemental Figure S1).…”

Section: Cloning Verification and Transformation Of Cp3gt Into Pichia...mentioning

confidence: 99%

“…This modified plasmid was subsequently introduced into competent Pichia pastoris cells via electroporation, as previously described [49]. To confirm the Cp3GT gene's successful integration into the Pichia genome, colony PCR was performed using 5 ′ and 3 ′ primers for the plasmid-encoded alcohol oxidase (AOX) gene.…”

Section: Cloning Verification and Transformation Of Cp3gt Into Pichia...mentioning

confidence: 99%

“…A flavonol-specific 3-O glucosyltransferase (Cp3GT, Genbank Accession ID-ACS15351.1) was identified in Citrus paradisi and shown through biochemical characterization to preferentially glucosylate the flavonols quercetin, kaempferol, myricetin, and fisetin at the 3-OH position [30,31]. This enzyme exhibits a unique specificity among glycosyltransferases, focusing exclusively on the 3-OH position of flavonols, a feature not reported in other GTs.…”

Section: Introductionmentioning

confidence: 99%

“…To counteract protease action, strains with reduced protease activity or those that are genetically modified to eliminate specific protease genes, such as PEP4, PRB1, and YPSs, are utilized [46]. Moreover, the adjustment of culture conditions and the integration of protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) have shown efficacy in minimizing protein degradation [30,31,43,47,48].…”

Section: Introductionmentioning

confidence: 99%

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Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi

Birchfield,

McIntosh

2024

BioTech

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Glycosyltransferases (GTs) are pivotal enzymes in the biosynthesis of various biological molecules. This study focuses on the scale-up, expression, and purification of a plant flavonol-specific 3-O glucosyltransferase (Cp3GT), a key enzyme from Citrus paradisi, for structural analysis and modeling. The challenges associated with recombinant protein production in Pichia pastoris, such as proteolytic degradation, were addressed through the optimization of culture conditions and purification processes. The purification strategy employed affinity, anion exchange, and size exclusion chromatography, leading to greater than 95% homogeneity for Cp3GT. In silico modeling, using D-I-TASSER and COFACTOR integrated with the AlphaFold2 pipeline, provided insights into the structural dynamics of Cp3GT and its ligand binding sites, offering predictions for enzyme–substrate interactions. These models were compared to experimentally derived structures, enhancing understanding of the enzyme’s functional mechanisms. The findings present a comprehensive approach to produce a highly purified Cp3GT which is suitable for crystallographic studies and to shed light on the structural basis of flavonol specificity in plant GTs. The significant implications of these results for synthetic biology and enzyme engineering in pharmaceutical applications are also considered.

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Section: Cloning Verification and Transformation Of Cp3gt Into Pichia...mentioning

confidence: 99%

Section: Cloning Verification and Transformation Of Cp3gt Into Pichia...mentioning

confidence: 99%

Section: Cloning Verification and Transformation Of Cp3gt Into Pichia...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi

Birchfield,

McIntosh

2024

BioTech

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The s-oph enzyme for efficient degradation of polyvinyl alcohol: soluble expression and catalytic properties

Wang,

Li,

Lin

et al. 2023

Mol Biol Rep

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BackgroundPolyvinyl alcohol (PVA) is one of the most widely used water-soluble polymers with great mechanical properties. However, water-soluble polymers are one of the major organic pollution sources in streams, river, and marine ecosystems. Once dispersed in aqueous systems, they can directly interfere with the life cycle of aquatic organisms due to their direct toxicity. Therefore, it is urgent to develop e cient microorganisms or enzyme to degrade it. The oxidized PVA hydrolase (OPHase) plays an important role in the pathway of PVA biodegradation. It is the key enzyme in the second step of PVA completely degradation. Methods and ResultsThe s-oph gene was cloned from laboratory isolated strain Sphingopyxis sp. M19. The s-oph gene was expressed in the E. coli system pET32a/s-oph expression vector in the form of an inclusion body. By binding with the molecular chaperone, pET32a/s-oph/BL21 (DE3)/pGro7 was constructed successfully, which enabled the s-oph gene to achieve soluble expression in E. coli. The s-oph gene expressed protein was puri ed at the yield of 16.8 mg L − 1 , and its catalytic activity reached 852.71 U mg − 1 . In the s-oph enzyme reaction system, the degradation e ciency of PVA can be increased to 233.5% compared to the controls. ConclusionThe s-oph enzyme had PVA degradation characteristics, high e ciency, speci city, and stability. The soph enzyme has good practical application potential in alleviating plastic pollution and protecting the environment.

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S-oph enzyme for efficient degradation of Polyvinyl alcohol: Soluble expression and Catalytic properties

Wang

Lin

et al. 2023

Preprint

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Background Polyvinyl alcohol (PVA) is one of the most widely used water-soluble polymers with great mechanical properties. However, water-soluble polymers are one of the major organic pollution sources in streams, river, and marine ecosystems. Once dispersed in aqueous systems, they can directly interfere with the life cycle of aquatic organisms due to their direct toxicity. Therefore, it is urgent to develop efficient microorganisms or enzyme to degrade it. The oxidized PVA hydrolase (OPHase) plays an important role in the pathway of PVA biodegradation. It is the key enzyme in the second step of PVA completely degradation. Methods and Results The s-oph gene was cloned from laboratory isolated strain Sphingopyxis sp. M19. The s-oph gene was expressed in the E. coli system pET32a/s-oph expression vector in the form of an inclusion body. By binding with the molecular chaperone, pET32a/s-oph/BL21 (DE3)/pGro7 was constructed successfully, which enabled the s-oph gene to achieve soluble expression in E. coli. The s-oph gene expressed protein was purified at the yield of 16.8 mg L− 1, and its catalytic activity reached 852.71 U mg− 1. In the s-oph enzyme reaction system, the degradation efficiency of PVA can be increased to 233.5% compared to the controls. Conclusion The s-oph enzyme had PVA degradation characteristics, high efficiency, specificity, and stability. The s-oph enzyme has good practical application potential in alleviating plastic pollution and protecting the environment.

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The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity

Cited by 4 publications

References 51 publications

Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi

Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi

The s-oph enzyme for efficient degradation of polyvinyl alcohol: soluble expression and catalytic properties

S-oph enzyme for efficient degradation of Polyvinyl alcohol: Soluble expression and Catalytic properties

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