Recombinant Oryza sativa subsp. japonica BURP domain-containing protein 9 (BURP9)

How is BURP9 structurally related to other BURP domain-containing proteins?

BURP9 shares structural homology with other BURP family members in rice. The characteristic BURP domain typically consists of several conserved motifs and a C-terminal region with repeated cysteine-histidine patterns. Based on ortholog analysis of related BURP proteins, the following structural relationships can be observed:

BURP9 would likely show similar domain architecture and evolutionary conservation patterns, as demonstrated for these related family members . Comparative sequence analysis across species reveals that BURP domains maintain highly conserved structural motifs despite sequence divergence.

What are the expression patterns of BURP9 in different tissues or developmental stages?

While specific expression data for BURP9 are not directly presented in the available research, methodological approaches used for other BURP proteins can be applied to characterize its expression:

• Employ RT-PCR and qRT-PCR to quantify relative expression across tissues (roots, shoots, leaves, panicles, and developing seeds) and under various stress conditions.

• Utilize RNA-Seq data from resources like Oryzabase to examine expression patterns across developmental stages .

• Develop promoter-reporter gene fusion constructs to visualize spatial and temporal expression patterns in transgenic rice.

• Perform in situ hybridization to precisely localize expression at the tissue and cellular levels.

Expression analysis of orthologous BURP domain proteins suggests tissue-specific patterns, with some members showing preferential expression in reproductive tissues while others respond to abiotic stress conditions.

What are the most effective methods for recombinant expression of BURP9?

Based on successful expression protocols for other recombinant rice proteins, the following methodological approach is recommended for BURP9:

• Expression System Selection:

  • E. coli is the most commonly used system, as demonstrated for recombinant Ory s 1 protein .

  • For complex proteins requiring post-translational modifications, consider Pichia pastoris or insect cell systems.

• Expression Optimization:

  • Add a His-tag for purification, following established protocols for rice proteins .

  • Test multiple expression conditions: temperature (18-37°C), induction periods (4-24 hours), and inducer concentrations.

  • Consider fusion partners (MBP, GST, SUMO) if solubility is problematic.

• Protein Specifications:

Codon optimization for the expression host may significantly improve yields, especially when expressing plant proteins in bacterial systems.

How can BURP9 be genetically modified to study its function?

Recent advances in rice transformation technologies enable multiple approaches for studying BURP9 function:

• CRISPR-Cas9 Gene Editing:

  • Design gRNAs targeting exonic regions of BURP9 to create knockout lines.

  • Employ base editing or prime editing for precise sequence modifications.

• Transformation Methods:

  • Utilize the immature-embryo method with Agrobacterium, which has demonstrated success across diverse Oryza species .

  • This approach has been effective for transforming multiple genome types (AA, BB, CC, BBCC, CCDD, FF, and HHJJ) .

• Expression Modifications:

  • Develop overexpression constructs using constitutive (CaMV 35S, Ubiquitin) or tissue-specific promoters.

  • Create fusion proteins with reporter tags (GFP, YFP) to track subcellular localization.

  • Implement inducible expression systems for temporal control.

• Protein Domain Analysis:

  • Generate truncated versions to identify functional domains.

  • Perform alanine scanning mutagenesis of conserved residues within the BURP domain.

The recently optimized Agrobacterium-mediated transformation protocols dramatically increase success rates for genetic modification of diverse rice varieties and related species .

What are the evolutionary relationships between BURP9 and other BURP domain-containing proteins?

Evolutionary analysis of BURP domain proteins reveals complex relationships across plant species:

• Orthologous Relationships:
  The InParanoid database identifies several ortholog groups for BURP proteins, with rice BURP domains showing conservation across diverse species:

• Phylogenetic Analysis:

  • BURP domain proteins likely originated before the divergence of monocots and dicots.

  • Gene duplication events have led to functional diversification within the BURP family.

  • Rice BURP proteins cluster into distinct subfamilies with specialized functions.

• Selective Pressure Analysis:

  • Examine non-synonymous to synonymous substitution ratios to identify domains under selection.

  • Compare sequences from wild Oryza accessions to assess natural variation and adaptation .

This evolutionary context provides crucial insights into BURP9's potential functional specialization and conservation across species.

How to design experiments to study BURP9 function in vivo?

A comprehensive experimental approach to characterize BURP9 function should include:

• Genetic Modification Strategy:

  • Generate CRISPR-Cas9 knockout lines targeting different exons of BURP9.

  • Create RNAi knockdown lines for partial suppression if complete knockout is lethal.

  • Develop overexpression lines using both constitutive and tissue-specific promoters.

  • Establish complementation lines by transforming knockout lines with native BURP9.

• Phenotypic Characterization:

  • Examine developmental phenotypes across all growth stages.

  • Assess responses to abiotic stresses (drought, salinity, temperature).

  • Analyze cellular and subcellular phenotypes using microscopy techniques.

  • Measure biochemical parameters related to cell wall composition if structural roles are suspected.

• Genetic Resources:

  • Utilize chromosome segment substitution lines (CSSLs) derived from wild Oryza species to study genetic interactions .

  • Consider monosomic alien addition lines (MAALs) that contain chromosomes from wild rice species in addition to O. sativa chromosomes for comparative functional analysis .

• Expression Analysis:

  • Perform tissue-specific and stress-responsive transcriptome analysis.

  • Use reporter gene fusions to visualize spatial and temporal expression patterns.

This multi-faceted approach will provide comprehensive insights into BURP9 function in rice biology.

What are the best approaches for purifying recombinant BURP9?

Purification of recombinant BURP9 requires a systematic approach to ensure high purity and biological activity:

• Initial Capture:

  • For His-tagged BURP9, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the primary method .

  • Optimize binding and elution conditions: test different imidazole concentrations (10-500 mM) for washing and elution.

• Secondary Purification:

  • Ion exchange chromatography based on BURP9's theoretical pI.

  • Size exclusion chromatography to remove aggregates and achieve >95% purity .

• Buffer Optimization:

  • Test stability in various buffers (pH 6.0-8.0).

  • Include stabilizing additives (glycerol, reducing agents) if needed.

  • Perform thermal shift assays to identify optimal buffer conditions.

• Quality Control:

• Storage Conditions:

  • Determine optimal storage conditions (-80°C, -20°C, 4°C).

  • Test stability with and without cryoprotectants.

  • Evaluate freeze-thaw stability.

These methodological considerations ensure production of high-quality BURP9 suitable for downstream functional and structural studies.

How to analyze the interaction partners of BURP9?

Identifying interaction partners is crucial for understanding BURP9's functional mechanisms. A comprehensive interactome analysis should include:

• In Vivo Approaches:

  • Co-immunoprecipitation (Co-IP) with anti-BURP9 antibodies followed by mass spectrometry.

  • Proximity-dependent biotin identification (BioID) by expressing BURP9-BirA fusion protein.

  • Fluorescence resonance energy transfer (FRET) for direct visualization of interactions in plant cells.

• In Vitro Methods:

  • Yeast two-hybrid screening against rice cDNA libraries.

  • Pull-down assays using recombinant BURP9 as bait.

  • Surface plasmon resonance (SPR) to measure binding kinetics.

• Bioinformatic Prediction:

  • Use interactome databases and protein-protein interaction prediction tools.

  • Analyze co-expression networks from transcriptomic data.

  • Identify proteins with complementary domains or motifs.

• Validation Strategy:

  • Confirm key interactions using multiple independent methods.

  • Perform domain mapping to identify interaction interfaces.

  • Test biological relevance through genetic analysis of interaction partners.

This systematic approach will reveal BURP9's position within cellular protein networks and provide insights into its molecular functions.

How to interpret contradictory results in BURP9 functional studies?

When faced with contradictory results in BURP9 research, apply this systematic approach:

• Experimental Variable Analysis:

  • Compare genetic backgrounds (japonica vs. indica cultivars).

  • Analyze developmental stages and tissue specificity.

  • Assess environmental conditions and stress treatments.

  • Evaluate methodological differences between studies.

• Redundancy Considerations:

  • Investigate potential functional redundancy with other BURP family members.

  • Examine expression profiles of all BURP proteins to identify co-expression patterns.

  • Consider creating multiple gene knockouts to overcome redundancy.

• Resolution Strategies:

  • Perform independent validation in standardized conditions.

  • Design experiments with appropriate positive and negative controls.

  • Collaborate with other laboratories for reproducibility assessment.

  • Conduct meta-analysis of all available data.

• Reconciliation Framework:

  • Develop models that accommodate seemingly contradictory results.

  • Consider context-dependent functions based on developmental stage or environmental conditions.

  • Propose testable hypotheses to resolve contradictions.

This methodical approach transforms contradictory results into opportunities for deeper mechanistic understanding of BURP9 function.

What bioinformatics tools are most appropriate for analyzing BURP9 sequence and structure?

A comprehensive bioinformatics analysis of BURP9 should employ multiple complementary approaches:

• Sequence Analysis:

  • Multiple sequence alignment: MUSCLE, Clustal Omega, T-Coffee

  • Motif identification: MEME, GLAM2

  • Domain prediction: InterPro, SMART, Pfam

  • Physicochemical property analysis: ProtParam, EMBOSS

• Structural Analysis:

  • Secondary structure prediction: PSIPRED, JPred

  • 3D structure prediction: AlphaFold2, I-TASSER, Phyre2

  • Protein disorder prediction: PONDR, IUPred

  • Molecular dynamics simulations: GROMACS, AMBER

• Evolutionary Analysis:

  • Phylogenetic tree construction: MEGA, PhyML, MrBayes

  • Selection pressure analysis: PAML, HyPhy

  • Ortholog identification: OrthoFinder, InParanoid

  • Synteny analysis: SynMap, MCScanX

• Rice-Specific Resources:

  • Oryzabase for genetic and genomic information

  • Rice Genome Annotation Project

  • SNP-Seek for polymorphism data

  • OryzaGenome for wild rice accessions

Integrating these diverse analytical approaches provides a comprehensive understanding of BURP9's molecular characteristics and evolutionary context.

How to compare BURP9 expression data across different experimental conditions?

Robust comparison of BURP9 expression across diverse experimental conditions requires:

• Data Normalization Strategies:

  • For RNA-Seq: TPM, FPKM, or rlog transformation

  • For microarray: RMA, VSN, or quantile normalization

  • For qRT-PCR: Selection of stable reference genes validated for specific conditions

• Statistical Analysis Framework:

  • Differential expression: DESeq2, edgeR (RNA-Seq); limma (microarray)

  • Multiple testing correction: Benjamini-Hochberg FDR

  • Effect size calculation: log2 fold change with confidence intervals

  • Power analysis to determine adequate sample sizes

• Visualization Methods:

  • Heat maps for multi-condition comparisons

  • Volcano plots for significance and magnitude assessment

  • Principal component analysis for pattern identification

  • Temporal expression profiles for time-course experiments

• Meta-Analysis Approach:

  • Standardized effect size calculation across studies

  • Random-effects models to account for inter-study variability

  • Sensitivity analysis to assess robustness of findings

This methodological framework enables rigorous comparison of BURP9 expression data, revealing consistent patterns across diverse experimental conditions.

What are common challenges in working with recombinant BURP9?

Researchers frequently encounter specific challenges when working with BURP domain-containing proteins:

• Expression Challenges:

  • Low expression levels due to codon bias or toxicity

  • Protein insolubility and inclusion body formation

  • Improper folding affecting functional activity

• Methodological Solutions:

  • Optimize codon usage for expression host

  • Test multiple fusion partners (MBP, GST, SUMO) for improved solubility

  • Reduce expression temperature (16-20°C) to enhance proper folding

  • Consider refolding protocols if inclusion bodies form

  • Express individual domains separately if full-length protein is problematic

• Purification Challenges:

  • Non-specific binding during affinity chromatography

  • Protein aggregation during concentration

  • Proteolytic degradation during purification

• Stability Considerations:

  • Test multiple buffer systems (HEPES, Tris, phosphate)

  • Evaluate stabilizing additives (glycerol, arginine, trehalose)

  • Optimize pH range (typically 6.5-8.0)

  • Include appropriate protease inhibitors

These methodological approaches address the specific biochemical challenges associated with recombinant BURP domain proteins.

How to validate antibody specificity for BURP9 detection?

Ensuring antibody specificity is critical for reliable BURP9 detection. A comprehensive validation strategy should include:

• Primary Validation Tests:

  • Western blot analysis comparing wild-type and BURP9 knockout/knockdown tissues

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

  • Pre-absorption tests with recombinant BURP9 protein

  • Peptide competition assays with immunizing peptides

• Cross-Reactivity Assessment:

  • Test against recombinant proteins of other BURP family members

  • Examine tissues with known expression profiles of BURP proteins

  • Perform immunodetection in heterologous systems expressing only BURP9

• Application-Specific Validation:

  • For immunohistochemistry: include appropriate negative controls and knockout tissues

  • For immunoprecipitation: verify enrichment by Western blot and mass spectrometry

  • For ELISA: establish detection limits and dynamic range

• Complementary Approaches:

  • Compare antibody results with GFP-tagged BURP9 localization

  • Validate with orthogonal detection methods (RNA expression, MS-based proteomics)

This rigorous validation ensures that experimental findings accurately reflect BURP9 biology rather than artifacts of non-specific antibody binding.

What controls should be included in BURP9 functional studies?

• Genetic Controls:

  • Wild-type plants of the same genetic background

  • Empty vector transformants for overexpression studies

  • CRISPR-Cas9 controls targeting non-coding regions

  • Complementation lines expressing BURP9 under native promoter

  • Multiple independent transgenic lines to control for positional effects

• Experimental Controls:

  • Time-course measurements to establish causality

  • Dose-response relationships for treatments

  • Internal standards for quantitative measurements

  • Technical and biological replicates (minimum n=3)

• Tissue-Specific Considerations:

  • Age-matched tissues for developmental studies

  • Circadian time-matched samples if BURP9 shows diurnal regulation

  • Appropriate stress controls for abiotic treatment studies

• Molecular Controls:

  • For protein-protein interaction: both positive and negative interaction controls

  • For transcriptional studies: stable reference genes validated for experimental conditions

  • For protein expression: loading controls and recombinant protein standards

These comprehensive control strategies ensure robust and reproducible findings in BURP9 functional studies.