Recombinant Pleuronectes platessa UDP-glucuronosyltransferase (ugt3)

What is Recombinant Pleuronectes platessa UDP-glucuronosyltransferase (ugt3)?

Recombinant Pleuronectes platessa UDP-glucuronosyltransferase (ugt3) is a full-length protein derived from the European plaice (Pleuronectes platessa). The protein is typically produced in E. coli expression systems and contains 472 amino acids (aa 1-472). For research applications, it is commonly available with a histidine tag to facilitate purification and detection .

UDP-glucuronosyltransferases are a family of enzymes involved in the conjugation of substrates with glucuronic acid, which typically enhances the water solubility of compounds and facilitates their excretion. This process is particularly important in detoxification pathways.

How is recombinant ugt3 produced for research applications?

The production of recombinant ugt3 from Pleuronectes platessa typically follows these methodological steps:

• Gene cloning and vector construction: The ugt3 gene sequence is amplified from Pleuronectes platessa tissue samples using PCR techniques with specific primers.

• Expression system selection: E. coli is the preferred host for expression of recombinant Pleuronectes platessa ugt3 as indicated in available research data .

• Vector design: Expression vectors incorporating histidine tags are commonly used to facilitate downstream purification processes.

• Protein expression: Following transformation of the expression vector into E. coli, culture conditions (temperature, induction timing, media composition) are optimized to maximize protein yield.

• Protein purification: Affinity chromatography using the histidine tag is employed for initial purification, potentially followed by additional chromatographic steps to achieve higher purity.

• Quality control: The final recombinant protein is assessed for purity, integrity, and activity using techniques such as SDS-PAGE, western blotting, and enzyme activity assays.

What are the standard applications of recombinant ugt3 in research settings?

Recombinant Pleuronectes platessa ugt3 is commonly employed in various research applications:

• Enzymatic activity studies: Characterizing substrate specificity and reaction kinetics of the glucuronidation process.

• Comparative biochemistry: Studying evolutionary differences in detoxification mechanisms across fish species.

• Environmental toxicology: Investigating the role of ugt3 in detoxifying environmental pollutants in marine organisms.

• Structure-function relationship analysis: Identifying critical domains for catalytic activity through mutagenesis studies.

• Drug metabolism studies: Examining the potential role of fish ugt3 in biotransformation of pharmaceutical compounds in aquatic environments.

How does ugt3 interact with other proteins in metabolic pathways?

Pleuronectes platessa ugt3 participates in several biochemical pathways, though comprehensive interaction data is still emerging in the literature. Researchers investigating ugt3 interactions should consider:

• Interaction partner identification approaches:

  • Protein co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening

  • Protein cross-linking studies

  • Pull-down assays using the recombinant His-tagged ugt3 as bait

• Pathway analysis methodologies:

  • Integration of transcriptomic and proteomic data

  • Metabolic flux analysis

  • Enzyme activity coupling assays

The available literature indicates that ugt3 likely participates in multiple metabolic pathways, though these pathways have not been fully characterized . A systematic approach to identifying interaction partners would significantly advance understanding of ugt3's role in Pleuronectes platessa metabolism.

What genetic variation exists in ugt3 genes across plaice populations?

The analysis of genetic variation in ugt3 genes across plaice populations can provide insights into evolutionary adaptations to different environmental conditions. Based on population genetics studies of Pleuronectes platessa:

• Population structuring and differentiation:

  • Significant genetic differentiation has been observed between Icelandic and continental European plaice populations (θ = 0.0290*** to 0.0456***) .

  • This suggests potential differences in ugt3 alleles between these geographically separated populations.

• Heterozygosity patterns:

  • Plaice populations consistently display heterozygote deficiencies .

  • Mean expected heterozygosity across microsatellite loci ranges from 0.721 to 0.770 , suggesting potentially high genetic diversity in functional genes like ugt3 as well.

• Methodological approaches for ugt3 variation analysis:

  • PCR amplification of ugt3 coding regions using primers designed from conserved regions

  • Next-generation sequencing to identify polymorphisms

  • Functional characterization of variant alleles through recombinant expression and activity assays

What experimental considerations are important when assessing ugt3 enzymatic activity?

When designing experiments to assess the enzymatic activity of recombinant Pleuronectes platessa ugt3, researchers should consider:

• Buffer composition optimization:

  • pH range (typically 7.0-8.0)

  • Ionic strength

  • Presence of divalent cations (Mg²⁺, Ca²⁺)

  • Reducing agents (DTT, β-mercaptoethanol)

• Substrate selection and preparation:

  • Natural vs. synthetic substrates

  • Substrate solubility considerations

  • Concentration ranges for kinetic studies

• Cofactor requirements:

  • UDP-glucuronic acid purity and concentration

  • Potential need for additional cofactors

• Detection methods:

  • Spectrophotometric assays

  • HPLC analysis of reaction products

  • Mass spectrometry for product identification

  • Radiometric assays using labeled substrates

• Controls and validation:

  • Heat-inactivated enzyme controls

  • Known ugt inhibitors as negative controls

  • Reference substrates with established kinetic parameters

How can researchers address the challenges of protein instability in ugt3 studies?

Recombinant membrane-associated enzymes like ugt3 often present stability challenges. Researchers can employ several strategies to overcome these issues:

• Storage condition optimization:

  • Assess protein stability at various temperatures (-80°C, -20°C, 4°C)

  • Evaluate cryoprotectant additives (glycerol, sugars)

  • Determine optimal protein concentration for storage

• Buffer formulation for stability enhancement:

  • Addition of detergents at concentrations below critical micelle concentration

  • Incorporation of stabilizing agents (glycerol, BSA)

  • Antioxidants to prevent oxidative damage

• Structural modifications approach:

  • Creation of fusion proteins with stability-enhancing partners

  • Directed evolution for improved stability

  • Structure-based design of stabilizing mutations

• Experimental design considerations:

  • Minimize freeze-thaw cycles

  • Prepare fresh enzyme preparations for critical experiments

  • Include time-course stability controls

How does genetic inbreeding impact ugt3 expression and function in plaice populations?

Research on Pleuronectes platessa populations has revealed evidence of inbreeding that may affect ugt3 and other genes:

• Inbreeding indicators in plaice populations:

  • Significant heterozygote deficiencies have been consistently observed with FIS estimates ranging from 0.151 to 0.233 .

  • Six out of eight microsatellite loci showed significant single-locus FIS estimates in >80% of studied cohorts .

  • Analysis of multilocus heterozygosity (MLH) showed higher frequency of lower MLH classes than expected under random mating (P<0.001) .

• Methodological approaches to study inbreeding effects on ugt3:

  • Quantitative PCR to measure expression levels in individuals with different inbreeding coefficients

  • Enzyme activity assays to correlate function with heterozygosity

  • Targeted sequencing of ugt3 alleles to identify functional polymorphisms

• Potential consequences for ugt3 function:

  • Reduced allelic diversity may limit adaptive capacity of detoxification systems

  • Expression levels may be affected by homozygosity at regulatory loci

  • Functional constraints due to fixation of suboptimal alleles

The observation that "low effective population size, in combination with fisheries related depletion of kin-structured spawning aggregations, may have led to inbreeding in plaice" suggests that detoxification pathways involving ugt3 may be under genetic constraint in some populations.

What are the comparative characteristics of ugt3 across different fish species?

When conducting comparative analyses of ugt3 across fish species, researchers should consider:

• Phylogenetic analysis approach:

  • Sequence alignment of ugt3 orthologs

  • Construction of phylogenetic trees to determine evolutionary relationships

  • Identification of conserved catalytic and substrate-binding domains

• Functional divergence assessment:

  • Recombinant expression of ugt3 orthologs from multiple species

  • Comparative substrate specificity profiling

  • Kinetic parameter determination (Km, Vmax, kcat)

• Structural comparison methodology:

  • Homology modeling based on available crystal structures

  • Molecular docking studies with various substrates

  • Molecular dynamics simulations to assess dynamic properties

• Ecological and environmental context:

  • Correlation of ugt3 properties with species habitat and exposure to xenobiotics

  • Assessment of selective pressures on ugt3 genes

  • Adaptation to specific environmental toxins

What are the optimal conditions for expressing recombinant Pleuronectes platessa ugt3 in E. coli?

Based on established protocols for recombinant protein expression in E. coli:

• Expression strain selection:

  • BL21(DE3) and derivatives for high-level expression

  • Rosetta strains for rare codon optimization

  • Origami strains for disulfide bond formation

• Vector and promoter considerations:

  • pET vectors with T7 promoter for high-level expression

  • tac or lac promoters for more controlled expression

  • Incorporation of His-tag for purification

• Induction parameter optimization:

  • IPTG concentration (typically 0.1-1.0 mM)

  • Induction temperature (often reduced to 16-25°C for membrane proteins)

  • Induction timing (mid-log phase, OD600 ~0.6-0.8)

  • Duration of induction (4-24 hours)

• Media formulation:

  • Rich media (LB, TB, 2YT) for biomass generation

  • Supplementation with glucose for catabolite repression

  • Addition of rare nutrients or cofactors if required

• Harvest and extraction considerations:

  • Cell lysis methods (sonication, homogenization, chemical lysis)

  • Inclusion body solubilization and refolding strategies if needed

  • Membrane fraction isolation protocols for membrane-associated forms

What analytical techniques are most effective for characterizing recombinant ugt3 structure and function?

A comprehensive characterization of recombinant Pleuronectes platessa ugt3 requires multiple analytical approaches:

• Structural characterization methods:

  • Circular dichroism spectroscopy for secondary structure assessment

  • Fluorescence spectroscopy for tertiary structure analysis

  • Limited proteolysis coupled with mass spectrometry for domain mapping

  • X-ray crystallography or cryo-EM for high-resolution structure determination

• Functional analysis techniques:

  • Spectrophotometric assays for real-time activity monitoring

  • HPLC or LC-MS/MS for product characterization

  • Isothermal titration calorimetry for binding studies

  • Surface plasmon resonance for interaction kinetics

• Quality assessment approaches:

  • Size-exclusion chromatography for oligomeric state determination

  • Dynamic light scattering for homogeneity analysis

  • Mass spectrometry for accurate mass determination and post-translational modifications

  • Thermal shift assays for stability assessment

The full-length recombinant Pleuronectes platessa ugt3 (aa 1-472) produced with His-tag in E. coli systems provides an excellent starting point for these characterization studies.

Key Properties of Recombinant Pleuronectes platessa ugt3

Population Genetic Parameters in Pleuronectes platessa

PCR Amplification Parameters for Pleuronectes platessa Genetic Analysis

What emerging technologies will advance ugt3 research?

Several cutting-edge approaches are poised to enhance our understanding of Pleuronectes platessa ugt3:

• CRISPR-Cas9 genome editing:

  • Generation of ugt3 knockout or modified fish models

  • Introduction of reporter tags at endogenous loci

  • Precise manipulation of regulatory elements

• Single-cell transcriptomics and proteomics:

  • Cell-specific expression patterns of ugt3

  • Identification of co-expressed genes

  • Regulatory network mapping

• Structural biology advances:

  • AlphaFold2 or RoseTTAFold prediction of ugt3 structure

  • Cryo-EM for membrane-associated forms

  • Time-resolved structural studies during catalysis

• Systems biology integration:

  • Multi-omics data integration for pathway mapping

  • Mathematical modeling of detoxification networks

  • In silico prediction of substrate profiles