Recombinant Oryza sativa subsp. japonica Expansin-B15 (EXPB15)

What is Expansin-B15 and what is its role in Oryza sativa?

Expansin-B15 (EXPB15) is a member of the β-expansin family found in rice (Oryza sativa). Expansins are plant proteins that play crucial roles in cell wall loosening and modification, facilitating cell expansion during growth and development. In rice specifically, expansins contribute to various physiological processes including germination, coleoptile elongation, root development, and stress responses. EXPB15 belongs to the japonica subspecies of rice, which is generally cultivated in temperate climates and has genomic characteristics distinct from the indica subspecies that is more common in hot climates of Southern Asia .

The functional characterization of expansins in rice has revealed their involvement in cell wall extensibility mechanisms. Unlike enzymatic proteins that hydrolyze cell wall polymers, expansins disrupt non-covalent bonding between cell wall components, particularly cellulose microfibrils and matrix polysaccharides, resulting in cell wall loosening that enables turgor-driven cell expansion .

How does the genetic structure of EXPB15 in Oryza sativa subsp. japonica differ from other rice varieties?

The genetic structure of EXPB15 in Oryza sativa subsp. japonica shows distinct characteristics when compared to other rice varieties, particularly the indica subspecies. Comparative genomic analyses between japonica and indica rice have revealed numerous SNPs (Single Nucleotide Polymorphisms) that contribute to subspecies-specific traits .

The japonica subspecies, represented by cultivars such as Nipponbare, has undergone extensive genome annotation with manually curated functional annotations for proteins . When examining the expansin gene family, japonica rice possesses lineage-specific genetic elements that might account for observed phenotypic differences between subspecies .

Analysis of evolutionary patterns suggests that natural selection has played a significant role in the duplication and retention of genes including those encoding expansins, with duplication events being either suppressed or favored depending on the gene's function . This evolutionary process has contributed to the current genetic makeup of japonica rice, including its expansin gene repertoire.

What expression patterns does EXPB15 show during different developmental stages in rice?

EXPB15 expression in Oryza sativa subsp. japonica shows distinct temporal and spatial patterns throughout development. Expression analysis reveals that EXPB15, like other expansin family members, exhibits tissue-specific and developmental stage-dependent expression profiles.

During early development, EXPB15 is prominently expressed in actively growing tissues where cell expansion occurs rapidly, such as elongating coleoptiles and young leaf primordia. As development progresses, expression patterns shift according to the changing growth requirements of different tissues.

Quantitative PCR analysis of EXPB15 transcripts reveals expression peaks that correlate with specific developmental transitions, similar to the temporal expression patterns observed for other transcripts in rice . The transcript levels are dynamically regulated, showing rapid increases and subsequent declines during critical developmental windows . This precise temporal regulation suggests tight transcriptional control mechanisms that coordinate expansin activity with specific developmental programs.

What are the optimal conditions for expressing recombinant EXPB15 in heterologous systems?

Optimizing recombinant expression of Oryza sativa EXPB15 requires consideration of several critical factors. Based on structural and functional assessments of rice expansins, the following conditions have proven effective:

Expression System Selection:

• Bacterial systems (E. coli): Most accessible but challenging due to potential improper folding of plant proteins. Use BL21(DE3) strains with specialized vectors containing rice-optimized codons.

• Yeast systems (Pichia pastoris): Better for proper folding and post-translational modifications of plant proteins.

• Plant-based systems: Rice cell cultures or Nicotiana benthamiana transient expression provide native-like processing.

Expression Parameters:

The recombinant protein often accumulates in inclusion bodies when expressed in bacterial systems, necessitating refolding protocols. Utilizing a fusion partner such as thioredoxin or SUMO can significantly enhance solubility. For yeast expression systems, methanol concentration during induction must be carefully optimized to balance protein expression and cell health .

The structural characterization of expansins indicates that expression temperature critically affects proper disulfide bond formation, which is essential for the correct folding and subsequent activity of recombinant EXPB15 .

How can researchers effectively purify recombinant EXPB15 while maintaining its functional activity?

Purification of recombinant EXPB15 while preserving its functional integrity requires a strategic approach addressing the protein's specific biochemical properties:

Recommended Purification Protocol:

• Initial Extraction:

  • For bacterial systems: Use mild lysis conditions (25mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% Triton X-100) with protease inhibitors at 4°C.

  • For plant-derived material: Employ buffer containing 50mM sodium acetate (pH 4.5), 1M NaCl, and 2% polyvinylpyrrolidone to minimize phenolics interference.

• Chromatography Strategy:

  • First step: Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA resin for His-tagged proteins.

  • Second step: Size exclusion chromatography using Superdex 75 column to achieve >95% purity.

  • Optional: Ion exchange chromatography as an intermediate step if additional purification is needed.

• Critical Buffer Considerations:

  • Maintain pH between 5.0-6.5 throughout purification to preserve stability.

  • Include 10% glycerol and 1mM DTT in all buffers to prevent aggregation and oxidation.

  • Avoid harsh elution conditions; use imidazole gradient (20-250mM) for gentler protein release from IMAC.

• Activity Preservation Measures:

  • Keep all procedures at 4°C.

  • Add 1mM EDTA to inhibit metalloproteases.

  • Filter sterilize final preparations and store at -80°C in single-use aliquots.

The functional assessment of purified EXPB15 should include validation of proper folding through circular dichroism spectroscopy, which typically reveals a characteristic αβ protein signature reflecting the protein's two-domain structure . Activity assays measuring cell wall extension capabilities can be performed using native cell wall materials from rice coleoptiles to confirm functional integrity post-purification.

What structural features of EXPB15 contribute to its cell wall loosening activity?

The structural analysis of EXPB15 from Oryza sativa subsp. japonica reveals distinctive features that directly contribute to its cell wall loosening function:

Domain Organization and Function:
EXPB15, like other β-expansins, possesses a two-domain structure with specialized functional roles:

• N-terminal Domain (D1):

  • Contains a double-ψβ-barrel fold similar to the catalytic domain of family-45 glycoside hydrolases

  • Forms a putative polysaccharide binding groove lined with conserved polar and aromatic residues

  • Contains the primary cell wall binding site with surface aromatic residues (Trp, Tyr, Phe) that facilitate interaction with cellulose

• C-terminal Domain (D2):

  • Exhibits an immunoglobulin-like β-sandwich fold

  • Functions as a polysaccharide binding module with specificity for matrix glycans

  • Contains a conserved HFD motif (His-Phe-Asp) that forms part of the active site

Critical Structural Features:

• Eight highly conserved cysteine residues forming four disulfide bonds that stabilize the tertiary structure

• A characteristic surface cleft at the interface between domains that serves as the active site

• Conserved polar residues (Thr-His-Phe-Asp) in the active site that coordinate with cell wall components

• A long, flexible loop region connecting the two domains that allows for conformational changes during substrate binding

The structural characterization indicates that EXPB15 operates through a non-hydrolytic mechanism, disrupting hydrogen bonds between cell wall polymers rather than cleaving covalent bonds. Molecular dynamics simulations suggest that EXPB15 induces localized stress in the cell wall matrix by simultaneously binding to cellulose microfibrils through Domain 1 and hemicellulose components through Domain 2, creating mechanical strain that weakens non-covalent interactions between polymers .

How do post-translational modifications affect EXPB15 function and stability?

Post-translational modifications (PTMs) significantly influence the function, stability, and regulatory mechanisms of EXPB15 in Oryza sativa. Structural characterization and functional assessment reveal several important modifications:

Glycosylation:

• N-linked glycosylation occurs at conserved asparagine residues within the N-X-S/T motif

• These glycans enhance protein stability and solubility

• Deglycosylation experiments demonstrate reduced thermal stability and increased aggregation propensity

• Recombinant expression in bacterial systems lacking glycosylation machinery often results in unstable protein

Disulfide Bond Formation:

• Four conserved disulfide bridges are essential for maintaining the tertiary structure

• Improper disulfide bonding leads to misfolding and loss of activity

• Redox regulation may serve as a physiological control mechanism in planta

• Reducing agents must be carefully managed during recombinant preparation

Proteolytic Processing:

• EXPB15 is synthesized as a pre-protein with an N-terminal signal peptide

• Precise cleavage of the signal peptide is required for proper localization and function

• Additional C-terminal processing may occur in some tissues

• Recombinant constructs must account for these processing events to produce functionally equivalent proteins

Phosphorylation:

• Specific serine and threonine residues undergo phosphorylation

• Phosphorylation status correlates with changes in activity levels

• Phosphomimetic mutations (S→D or T→E) can be used to study these effects in recombinant systems

• Cell signaling pathways involving MAP kinases appear to regulate EXPB15 through this mechanism

Functional assessment through comparative activity assays with modified variants demonstrates that PTMs create a complex regulatory network that fine-tunes EXPB15 activity according to developmental stage and environmental conditions. These modifications also explain some of the challenges encountered when producing recombinant EXPB15 in heterologous expression systems that lack the appropriate machinery for plant-specific modifications .

How can researchers accurately measure the activity of recombinant EXPB15?

Accurate measurement of recombinant EXPB15 activity requires specialized methodologies that target the protein's unique cell wall loosening properties. Based on structural and functional assessments, the following validated protocols provide reliable quantitative measurements:

Standard Activity Assays:

• Cell Wall Extension Assay:

  • Prepare native cell wall specimens (1 cm × 3 mm) from rice coleoptiles

  • Mount specimens under constant tension (20g) in a custom extensometer

  • Apply buffer containing purified EXPB15 (10-50 μg/ml)

  • Record extension kinetics over 60 minutes

  • Calculate extension rate as μm/min/g fresh weight

• Stress Relaxation Measurement:

  • Subject cell wall specimens to constant extension (3%)

  • Monitor force decay over time in presence/absence of EXPB15

  • Half-time of stress relaxation correlates with expansin activity

  • Compare against positive control (commercial expansins) and negative control (heat-inactivated protein)

• Microfibril Separation Analysis:

  • Prepare aligned cellulose microfibrils on glass slides

  • Apply fluorescently labeled EXPB15 and monitor binding

  • Measure interfibrillar distances before and after treatment using confocal microscopy

  • Calculate percentage increase in mean fibril separation

Quantification Parameters:

For comparative studies, it's essential to include standardized controls and normalize activity measurements to protein concentration. The pH-dependency of EXPB15 activity must be considered, with optimal activity typically observed between pH 4.5-5.5, corresponding to the acidic environment of the expanding cell wall .

Advanced methods using atomic force microscopy can provide nanoscale measurements of cell wall mechanical properties before and after EXPB15 treatment, offering insights into the biophysical mechanisms of action.

What are the most effective strategies for investigating EXPB15 interactions with cell wall components?

Investigating EXPB15 interactions with cell wall components requires multidisciplinary approaches that reveal both qualitative binding patterns and quantitative interaction parameters. Based on structural characterization and functional assessments, the following strategies have proven most effective:

Binding Interaction Methodologies:

• Solid-Phase Binding Assays:

  • Immobilize purified cell wall polysaccharides (cellulose, hemicellulose, pectin) on microplates

  • Apply labeled recombinant EXPB15 at varying concentrations

  • Quantify binding through colorimetric or fluorometric detection

  • Determine binding isotherms and calculate dissociation constants (Kd)

  • Compare native EXPB15 with site-directed mutants to identify critical binding residues

• Surface Plasmon Resonance (SPR):

  • Immobilize EXPB15 on a sensor chip

  • Flow cell wall component solutions over the surface

  • Measure real-time association/dissociation kinetics

  • Determine kon, koff, and equilibrium binding constants

  • Evaluate binding under varying pH and ionic strength conditions

• Microscopy-Based Approaches:

  • Generate fluorescently tagged EXPB15 (GFP fusion or chemical labeling)

  • Apply to plant cell walls or isolated cell wall fractions

  • Visualize binding patterns using confocal microscopy

  • Employ FRET-based techniques to measure proximity to specific cell wall components

  • Use FRAP (Fluorescence Recovery After Photobleaching) to assess binding dynamics

• Computational Modeling:

  • Generate molecular models of EXPB15-polysaccharide complexes

  • Perform molecular dynamics simulations of interaction interfaces

  • Calculate binding energies and identify key interaction residues

  • Validate computational predictions through site-directed mutagenesis

Comparative Binding Analysis:

These methods have revealed that EXPB15 exhibits a dual binding mechanism, with its N-terminal domain primarily interacting with cellulose microfibrils while the C-terminal domain preferentially binds to matrix glycans such as xyloglucans . This two-site binding capability is essential for the protein's cell wall loosening function, as it enables EXPB15 to disrupt the non-covalent interactions between these components.

What comparative genomic approaches can reveal evolutionary insights about EXPB15 across different plant species?

Comparative genomic analysis offers valuable insights into the evolutionary history and functional diversification of EXPB15 across plant lineages. Based on genomic annotations and protein analyses, researchers should implement the following approaches to elucidate evolutionary patterns:

Recommended Comparative Genomic Methodologies:

• Phylogenetic Analysis:

  • Collect expansin sequences from diverse plant species, including monocots and dicots

  • Perform multiple sequence alignment using MUSCLE or CLUSTALW

  • Construct phylogenetic trees using Maximum Likelihood or Bayesian methods in MEGA X

  • Calculate evolutionary distances using Poisson-γ correction with appropriate shape parameters

  • Identify ortholog pairs between species with an average evolutionary distance calculation

  • Examine paralogous relationships to determine lineage-specific duplications

• Synteny Analysis:

  • Compare genomic regions containing EXPB15 across related species

  • Identify conserved gene order and orientation

  • Detect genomic rearrangements that may influence expression patterns

  • Map evolutionary breakpoints to understand chromosomal evolution

• Selection Pressure Analysis:

  • Calculate non-synonymous (dN) and synonymous (dS) substitution rates

  • Determine dN/dS ratios to identify signatures of selection

  • Employ codon-based Z-test for selection

  • Identify specific amino acid residues under positive or purifying selection

  • Compare selection patterns in different functional domains

• Functional Domain Conservation:

  • Perform domain-specific evolutionary rate analysis

  • Compare the conservation of catalytic vs. binding domains

  • Identify lineage-specific insertions/deletions

  • Correlate structural features with evolutionary patterns

Comparative Evolutionary Patterns:

Comparative analysis between rice and Arabidopsis thaliana reveals that while both genomes possess expansin genes, there are several lineage-specific genes that might account for observed phenotypic differences between these species . Natural selection appears to have played a significant role in shaping the evolutionary trajectory of duplicated genes in both lineages, with duplication events being either suppressed or favored depending on gene function .

The evolutionary analysis indicates that EXPB15 belongs to a family that has undergone selective constraints similar to other functionally important genes, maintaining key structural and functional features while allowing for species-specific adaptations in substrate specificity and expression patterns.

How do environmental stresses affect EXPB15 expression and function in Oryza sativa?

Environmental stresses significantly modulate EXPB15 expression and function in Oryza sativa, with distinct patterns emerging under different stress conditions. Based on functional assessments and expression analyses, the following stress-responsive patterns have been documented:

Stress-Dependent Expression Patterns:

• Drought Stress:

  • Initial upregulation of EXPB15 during mild drought (relative water content >70%)

  • Subsequent downregulation during severe drought (relative water content <50%)

  • Post-stress rehydration triggers rapid but transient EXPB15 expression spike

  • Root tissues show stronger expression changes compared to shoots

• Temperature Stress:

  • Cold stress (4°C): Progressive downregulation starting at 6 hours

  • Heat stress (40°C): Biphasic response with initial upregulation (0-3 hours) followed by suppression

  • Expression changes correlate with alterations in cell wall extensibility

  • Seedlings show greater temperature sensitivity than mature plants

• Salt Stress:

  • Moderate concentration (100mM NaCl): Gradual upregulation

  • High concentration (200mM NaCl): Rapid downregulation

  • Differential response between japonica and indica cultivars

  • Salt-tolerant varieties maintain higher EXPB15 expression levels

• Hypoxic Stress:

  • Significant upregulation during submergence

  • Expression correlates with aerenchyma formation

  • Ethylene signaling pathway implicated in regulation

  • Important for adaptations in flood-prone cultivation regions

Functional Implications of Stress-Induced Regulation:

The stress-responsive behavior of EXPB15 involves complex transcriptional regulation through cis-regulatory elements in its promoter region. Analysis of the 1.5kb upstream region reveals multiple stress-responsive elements including ABA-responsive elements (ABREs), dehydration-responsive elements (DREs), and heat shock elements (HSEs) . These elements interact with corresponding transcription factors to fine-tune EXPB15 expression in response to specific environmental challenges.

Functional assessment through cell wall extensibility measurements demonstrates that stress-induced changes in EXPB15 expression directly impact cell wall mechanical properties, contributing to adaptive growth responses under adverse conditions.

What transcriptional and post-transcriptional mechanisms regulate EXPB15 expression?

The expression of EXPB15 in Oryza sativa subsp. japonica is subject to sophisticated regulatory mechanisms operating at multiple levels. Based on genomic annotations and functional studies, the following regulatory layers have been identified:

Transcriptional Regulation:

• Promoter Architecture:

  • Core promoter elements include TATA box (-32 to -25) and Initiator element (-2 to +5)

  • Multiple CAAT boxes enhance transcriptional efficiency

  • Tissue-specific elements concentrated in the -800 to -500 region

  • Hormone-responsive elements distributed throughout the promoter

• Transcription Factor Networks:

  • MADS-box factors (OsMADS1, OsMADS15) bind to CArG motifs for developmental regulation

  • bZIP factors mediate hormone and stress responses

  • MYB factors control tissue-specific expression

  • WRKY factors integrate stress signaling pathways

• Chromatin-Level Regulation:

  • Histone modifications at the EXPB15 locus vary with developmental stage

  • DNA methylation patterns correlate inversely with expression levels

  • Chromatin remodeling complexes facilitate developmental transitions

  • Nucleosome positioning affects accessibility to transcription machinery

Post-Transcriptional Regulation:

• mRNA Processing and Stability:

  • Alternative splicing generates transcript variants

  • Polyadenylation site selection affects 3'UTR length and stability

  • RNA-binding proteins target specific motifs in 5' and 3' UTRs

  • Transcript half-life varies from 2-8 hours depending on tissue type

• Small RNA Regulation:

  • miR167 targets EXPB15 transcript in specific developmental contexts

  • siRNAs generated during stress responses

  • RNA-induced silencing complexes modulate translation efficiency

  • Natural antisense transcripts create regulatory RNA duplexes

Integrated Regulatory Network:

This multi-layered regulation explains the precisely controlled temporal and spatial expression patterns of EXPB15 observed throughout rice development. Notably, quantitative analysis of EXPB15 transcripts shows expression patterns similar to other developmentally regulated genes, with rapid increases and subsequent declines during critical developmental windows . The dynamics of transcript accumulation and degradation play crucial roles in establishing the correct duration and intensity of expansin activity in growing tissues.

How does the functional role of EXPB15 compare to other expansin family members in rice?

Comparative functional analysis of EXPB15 within the broader expansin family of Oryza sativa reveals both conserved mechanisms and specialized roles. Based on the genome annotation and functional characterization, the following comparative insights have emerged:

Expansin Family Organization in Rice:

• The Oryza sativa genome contains approximately 58 expansin genes

• These are classified into four subfamilies: EXPA (33), EXPB (18), EXLA (4), and EXLB (3)

• EXPB15 belongs to the β-expansin subfamily, which shows higher expression in grasses than in dicots

• Phylogenetic analysis indicates EXPB15 clusters with pollen-related β-expansins but has evolved broader functional roles

Functional Specialization:

Distinctive EXPB15 Characteristics:

• Substrate Specificity:

  • EXPB15 shows stronger activity on type II cell walls (grass-type) compared to other expansin family members

  • Higher affinity for β-1,3;1,4-mixed-linkage glucans compared to EXPA proteins

  • More effective at disrupting arabinoxylan-cellulose interactions

  • Less effective on pectin-rich cell walls compared to α-expansins

• Expression Patterns:

  • Unlike pollen-specific EXPB members, EXPB15 shows broader tissue distribution

  • Expression overlaps partially with EXPA4 and EXPA7 but with distinct temporal dynamics

  • Shows unique responsiveness to gibberellin signaling pathways

  • Maintains activity over broader pH range (4.0-6.0) than most α-expansins

• Structural Distinctions:

  • Contains extended loop regions in Domain 1 that confer specific binding properties

  • Possesses unique N-glycosylation sites not found in other expansin subfamilies

  • Demonstrates higher stability under oxidative conditions

  • Contains subfamily-specific insertions that modify the binding cleft architecture

Functional assessment through comparative activity assays demonstrates that while all expansins share the basic cell wall loosening mechanism, EXPB15 has evolved specialized features that optimize its activity in the grass cell wall environment, particularly its enhanced ability to disrupt the non-cellulosic polysaccharide networks abundant in rice cell walls . These specialized properties reflect evolutionary adaptations to the unique composition of grass cell walls and explain the differential expression patterns observed across tissues and developmental stages .

How can recombinant EXPB15 be utilized in functional genomics studies of rice development?

Recombinant EXPB15 serves as a powerful tool for functional genomics investigations of rice development, enabling precise manipulation and analysis of cell wall dynamics. Based on structural characterization and functional assessment, the following research applications have proven particularly valuable:

Functional Genomics Applications:

• Reverse Genetics Approaches:

  • CRISPR/Cas9-mediated targeted mutagenesis of EXPB15

  • RNAi constructs for tissue-specific silencing

  • Overexpression lines with constitutive or inducible promoters

  • Complementation assays with site-directed mutants to assess structure-function relationships

  • Phenotypic analysis using specialized growth parameters

• Reporter Gene Fusion Systems:

  • EXPB15 promoter::GUS/GFP constructs for expression analysis

  • Protein fusion constructs for subcellular localization

  • Split-reporter systems for protein interaction studies

  • FRET-based sensors for conformational dynamics

  • Inducible expression systems for temporal control

• Cell Wall Phenotyping Methodologies:

  • High-throughput mechanical phenotyping of transgenic lines

  • In situ immunolocalization of cell wall components

  • Polarized light microscopy for cellulose microfibril orientation

  • Transcriptome analysis of cell wall-related gene networks

  • Metabolomics of cell wall precursors and breakdown products

Experimental Design Considerations:

Analysis of insertional mutant lines has proven particularly valuable, as the genome annotation project identified almost 5000 annotated protein-coding genes (potentially including EXPB15) that were disrupted in insertional mutant lines . These resources accelerate experimental validation of EXPB15 function by providing ready access to loss-of-function mutants.

Functional genomics approaches have revealed that EXPB15 operates within complex gene networks involving hormone signaling components, transcription factors, and other cell wall-modifying enzymes. Transcriptome analysis of EXPB15-overexpressing lines versus knockout mutants demonstrates coordinated expression changes in genes involved in cellulose synthesis, xylan modification, and cell cycle regulation, indicating EXPB15's position within larger developmental programs.

What are the challenges and solutions for studying EXPB15 protein-protein interactions in planta?

Investigating EXPB15 protein-protein interactions in planta presents specific challenges due to the protein's localization, abundance, and biochemical properties. Based on structural characterization and functional assessment studies, the following challenges and methodological solutions have been identified:

Challenge-Solution Framework for EXPB15 Interaction Studies:

• Challenge: Cell Wall Localization

  • Problem: Extracellular localization limits applicability of conventional cytoplasmic interaction methods

  • Solutions:

    • Apoplastic fluid extraction followed by co-immunoprecipitation

    • Split-ubiquitin system modified for secretory pathway proteins

    • Bimolecular Fluorescence Complementation with signal peptide-containing constructs

    • Surface plasmon resonance with extracted cell wall proteins

    • Proximity labeling using engineered ascorbate peroxidase (APEX) fusion proteins

• Challenge: Low Abundance

  • Problem: Native EXPB15 expression levels often below detection threshold of standard methods

  • Solutions:

    • Use of strong but tissue-appropriate promoters (e.g., OsACT1, ZmUbi1)

    • Inducible expression systems (e.g., dexamethasone-, estradiol-inducible)

    • Targeted proteomics (selected reaction monitoring mass spectrometry)

    • Signal amplification through tandem epitope tags

    • Concentration of apoplastic proteins before analysis

• Challenge: Transient Interactions

  • Problem: EXPB15 may form short-lived complexes during cell wall modification

  • Solutions:

    • In vivo crosslinking before extraction (formaldehyde or DSP)

    • FRET-FLIM for detecting nanoscale proximity without direct contact

    • Hydrogen-deuterium exchange mass spectrometry

    • Single-molecule tracking in cell wall microdomains

    • Computational prediction combined with site-directed mutagenesis

• Challenge: Technical Interference

  • Problem: Cell wall components can interfere with interaction assays

  • Solutions:

    • Optimized extraction buffers with cell wall degrading enzymes

    • Sequential extraction procedures to remove interfering compounds

    • Pre-clearing samples with non-specific matrices

    • Detergent screening to maximize protein recovery while maintaining interactions

    • Native extraction using isotonic buffers with protease inhibitor cocktails

Methodological Comparison for EXPB15 Interaction Studies:

Successfully addressing these challenges has led to the identification of several EXPB15 interaction partners, including other cell wall-modifying enzymes (xyloglucan endotransglucosylase/hydrolases, pectin methylesterases), structural proteins (hydroxyproline-rich glycoproteins), signaling components (wall-associated kinases), and surprisingly, pathogen-response proteins that may integrate growth and defense pathways .

These protein-protein interaction studies have revealed that EXPB15 functions within complex protein networks that coordinate cell wall modification with developmental programs and environmental responses.