Recombinant Phaseolus vulgaris 43 kDa cell wall protein

What is the Phaseolus vulgaris 43 kDa cell wall protein and what is its function?

The Phaseolus vulgaris (common bean) 43 kDa cell wall protein belongs to a class of structural proteins involved in cell wall integrity, defense responses, and potential signaling functions. While specific research on this particular protein is limited, proteomic studies of Phaseolus vulgaris have identified numerous proteins that participate in stress responses, particularly drought tolerance . Cell wall proteins often show differential expression under various stress conditions, suggesting their importance in plant adaptation mechanisms.

Methodology for function identification typically includes:

• Subcellular localization using fluorescent protein tagging

• Protein-protein interaction studies to identify binding partners

• Comparative analysis with homologous proteins in related species

• Gene knockout/knockdown studies to observe phenotypic effects

• Expression profiling under various biotic and abiotic stresses

What expression systems have been successfully used for recombinant production of Phaseolus vulgaris proteins?

For successful recombinant production of Phaseolus vulgaris proteins, researchers should consider the following expression systems:

• Bacterial systems (E. coli):

  • Advantages: Rapid growth, high yield, well-established protocols

  • Best for: Proteins without complex post-translational modifications

  • Methodology: Use of BL21(DE3) or Rosetta strains with pET vector systems

• Yeast systems (Pichia pastoris):

  • Advantages: Eukaryotic processing, protein secretion

  • Best for: Proteins requiring some post-translational modifications

  • Methodology: Integration of expression cassette into yeast genome

• Plant-based systems:

  • Advantages: Native-like environment, proper folding

  • Best for: Proteins requiring plant-specific modifications

  • Methodology: Transient expression in Nicotiana benthamiana or stable transformation in Arabidopsis

• Insect cell systems:

  • Advantages: Complex protein folding, higher eukaryotic modifications

  • Best for: Multi-domain proteins with disulfide bonds

  • Methodology: Baculovirus expression vectors in Sf9 or High Five cells

Transcriptome analysis of Phaseolus vulgaris has identified numerous expressed sequence tags (ESTs) that could be used to optimize codon usage for heterologous expression .

What purification techniques are most effective for isolating recombinant Phaseolus vulgaris proteins?

A systematic purification strategy for recombinant Phaseolus vulgaris proteins typically follows this methodology:

• Initial capture:

  • Affinity chromatography (His-tag, GST-tag)

  • Methodology: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins

• Intermediate purification:

  • Ion exchange chromatography

  • Methodology: Select cation/anion exchangers based on protein isoelectric point

• Fine purification:

  • Size exclusion chromatography

  • Methodology: Superdex or Sephacryl columns calibrated with molecular weight standards

Proteomic analysis methods described for Phaseolus vulgaris include sample preparation with DTT reduction, IAA alkylation, and trypsin digestion, which can be modified for protein purification workflows .

How can I verify the identity and purity of recombinant Phaseolus vulgaris 43 kDa cell wall protein?

Verification of identity and purity requires multiple complementary techniques:

• SDS-PAGE analysis:

  • Methodology: 12% polyacrylamide gels stained with Coomassie blue

  • Expected outcome: Single band at 43 kDa, purity >95%

• Western blotting:

  • Methodology: Transfer to PVDF membrane, probing with anti-tag antibody or custom antibody

  • Expected outcome: Specific recognition of target protein

• Mass spectrometry:

  • Methodology: In-gel tryptic digestion followed by LC-MS/MS

  • Expected outcome: Peptide coverage >80% of the predicted sequence

• N-terminal sequencing:

  • Methodology: Edman degradation of purified protein

  • Expected outcome: First 10-15 amino acids match predicted sequence

• Activity assays:

  • Methodology: Function-specific biochemical assays

  • Expected outcome: Activity comparable to native protein

Sample preparation protocols for proteomic analysis of Phaseolus vulgaris proteins include reduction with DTT and alkylation with IAA prior to trypsin digestion, which provides a foundation for mass spectrometry verification methods .

What are typical yields when expressing Phaseolus vulgaris proteins in heterologous systems?

Expression yields vary significantly depending on the system and specific protein:

Methodology for yield optimization:

• Expression screening: Test multiple constructs with different tags/fusion partners

• Culture optimization: Vary media composition, temperature, and induction parameters

• Scale-up strategy: Transition from shake flasks to bioreactors with controlled parameters

For Phaseolus vulgaris proteins, transcriptomic analysis has identified numerous ESTs that could inform construct design and expression optimization .

How can I design mutagenesis experiments to identify key functional domains in Phaseolus vulgaris 43 kDa cell wall protein?

Systematic mutagenesis requires a multi-step experimental approach:

• Computational analysis:

  • Methodology: Multiple sequence alignment across species using CLUSTAL Omega

  • Expected outcome: Identification of conserved residues and domains

• Structural prediction:

  • Methodology: AlphaFold or I-TASSER for 3D structure prediction

  • Expected outcome: Visualization of potential functional regions

• Mutagenesis strategy:

  • Alanine scanning: Replace conserved residues with alanine

  • Domain deletion: Remove entire predicted domains

  • Domain swapping: Exchange domains with related proteins

• Functional assessment:

  • Methodology: Compare wild-type and mutant proteins in activity assays

  • Statistical analysis: Minimum triplicate experiments with ANOVA

High-density SNP genotyping methods used for Phaseolus vulgaris genetic analysis provide insights into natural variation that can inform mutagenesis strategies .

What techniques are most effective for studying protein-protein interactions involving the Phaseolus vulgaris 43 kDa cell wall protein?

A comprehensive protein interaction study should employ multiple complementary techniques:

• In vitro methods:

  • Pull-down assays:
    Methodology: Immobilize tagged protein on resin, incubate with plant extract, elute and analyze bound proteins by mass spectrometry
    Expected outcome: Identification of direct binding partners

  • Surface plasmon resonance (SPR):
    Methodology: Immobilize protein on sensor chip, flow potential interactors, measure association/dissociation kinetics
    Expected outcome: Binding affinity (KD) and kinetic parameters

• In vivo methods:

  • Co-immunoprecipitation:
    Methodology: Generate antibodies against target protein, precipitate from plant extract, identify co-precipitated proteins
    Expected outcome: Physiologically relevant interaction partners

  • Bimolecular fluorescence complementation (BiFC):
    Methodology: Fuse protein pairs to split fluorescent protein halves, co-express in plant cells
    Expected outcome: Fluorescence restoration indicates interaction and localization

• High-throughput approaches:

  • Yeast two-hybrid screening:
    Methodology: Screen against cDNA library from Phaseolus vulgaris
    Expected outcome: Identification of binary interactions

  • Proximity-dependent biotin labeling:
    Methodology: Fuse BioID or TurboID to target protein, express in plant cells, identify biotinylated proteins
    Expected outcome: Spatial proteomics map of neighboring proteins

Proteomic approaches used to identify differentially expressed proteins in Phaseolus vulgaris can be adapted to detect and validate protein-protein interactions .

How does drought stress affect the expression and function of Phaseolus vulgaris cell wall proteins?

Based on proteomic analysis of drought responses in common bean, the following methodological approach is recommended:

• Experimental design:

  • Methodology: Compare tolerant genotypes (like SB-DT3 and SB-DT2) with sensitive genotypes (like Merlot and Stampede) under controlled drought conditions

  • Parameters: Soil moisture content, relative water content, photosynthetic rate

• Proteomic analysis:

  • Methodology: Protein extraction, digestion, and LC-MS/MS analysis

  • Data analysis: Label-free quantification, statistical testing for differential expression

• Functional classification:

  • Methodology: Gene Ontology enrichment analysis of differentially expressed proteins

  • Expected outcome: Identification of biological processes affected by drought

Proteomic studies have revealed that:

• Differentially expressed proteins (DEPs) are more abundant in drought-susceptible genotypes compared to tolerant lines

• Tolerant genotypes uniquely show DEPs related to sugar metabolism and plant signaling

• Sensitive genotypes display more DEPs involved in plant-pathogen interaction, proteasome function, and carbohydrate metabolism

• DEPs linked with chaperone function and signal transduction are significantly altered between tolerant and sensitive genotypes

How can SNP analysis be used to study variations in genes encoding Phaseolus vulgaris cell wall proteins?

SNP analysis for studying genetic variation involves these methodological steps:

• SNP discovery:

  • Whole-genome sequencing of diverse germplasm

  • Targeted resequencing of specific genes

  • Analysis of existing SNP arrays (e.g., 768-marker array or BARCBean6K_3 array)

• Genotyping approaches:

  • High-throughput SNP arrays

  • KASP (Kompetitive Allele Specific PCR) for targeted SNPs

  • Sequencing-based approaches (GBS, RAD-seq)

• Statistical analysis:

  • Linkage disequilibrium (LD) analysis across genomic regions

  • Population structure analysis to identify genepools

  • Haplotype construction and diversity analysis

• Functional implications:

  • QTL mapping to correlate SNPs with phenotypic traits

  • Prediction of SNP effects on protein structure and function

  • Development of marker-assisted selection strategies

Research on common bean has revealed:

• Uneven recombination rates across the genome (2.13 cM/Mb average)

• Recombination is highly repressed around centromeres and frequent outside peri-centromeric regions

• Stronger linkage disequilibrium within the Mesoamerican genepool compared to the Andean genepool

• SNP markers can track the introgression of specific traits like phaseolin and lectin deficiency

The methodology used in QTL analysis for sulfur amino acid traits in common bean provides a template for studying cell wall protein genetic variation, with high-density genetic maps allowing precise localization of traits .

What methods can be used to determine the crystal structure of recombinant Phaseolus vulgaris 43 kDa cell wall protein?

Determining the crystal structure requires a systematic approach:

• Protein preparation:

  • Methodology: Expression optimization for high yield and purity (>95% by SDS-PAGE)

  • Buffer screening: Test multiple buffers for optimal stability using differential scanning fluorimetry

• Crystallization screening:

  • Methodology: Vapor diffusion (hanging drop and sitting drop)

  • Initial screen: Commercial sparse matrix screens (Crystal Screen, PEG/Ion, Index)

  • Optimization: Fine-tuning promising conditions by varying precipitant concentration, pH, and additives

• Data collection:

  • Methodology: X-ray diffraction at synchrotron radiation facility

  • Expected resolution: Target <2.5 Å for detailed structural analysis

• Structure determination:

  • Molecular replacement: If homologous structures exist

  • Experimental phasing: Using heavy atom derivatives or selenomethionine-labeled protein

  • Model building and refinement: Iterative process using crystallographic software

• Alternative approaches if crystallization fails:

  • Construct optimization: Remove flexible regions identified by limited proteolysis

  • Surface entropy reduction: Mutate surface residues to enhance crystal contacts

  • Fusion partners: Use crystallization chaperones like T4 lysozyme

  • Alternative methods: Cryo-EM for larger assemblies or NMR for domains <25 kDa

How can I develop an assay to measure the enzymatic activity of recombinant Phaseolus vulgaris 43 kDa cell wall protein?

Developing a functional assay requires systematic investigation of potential enzymatic activities:

• Activity prediction:

  • Bioinformatic analysis: Sequence comparison with known enzymes

  • Structural analysis: Identification of potential catalytic sites

  • Literature review: Functions of homologous proteins

• Assay development strategy:

  • Substrate screening: Test panel of potential substrates based on predicted function

  • Reaction conditions: Optimize pH, temperature, buffer composition, cofactor requirements

  • Detection method: Spectrophotometric, fluorometric, or chromatographic approaches

• Validation and characterization:

  • Specificity controls: Heat-inactivated enzyme, catalytic site mutants

  • Kinetic analysis: Determine Km, Vmax, kcat parameters

  • Inhibitor studies: Test effect of potential inhibitors

For protein functionality assessment, in vitro protein digestibility methods using multi-enzyme solutions have been applied to Phaseolus vulgaris proteins, which could be adapted to assess the effect of cell wall proteins on digestibility .

What bioinformatic approaches can predict potential functions of uncharacterized domains in Phaseolus vulgaris proteins?

A comprehensive bioinformatic workflow includes:

• Sequence-based analysis:

  • Methodology: PSI-BLAST searches against non-redundant protein database

  • Expected outcome: Identification of remote homologs with known functions

  • Tools: HMMER searches against Pfam, InterPro, and SMART databases for domain identification

• Structure-based prediction:

  • Methodology: AlphaFold2 for ab initio 3D structure prediction

  • Expected outcome: Structural models with estimated accuracy

  • Analysis: Structural similarity searches using DALI or TM-align against PDB

• Functional site prediction:

  • Methodology: ConSurf for evolutionary conservation mapping

  • Expected outcome: Identification of functionally important residues

  • Tools: CASTp for pocket detection, SitePredict for ligand binding site prediction

• Integrated functional prediction:

  • Methodology: Gene Ontology term prediction using multiple tools

  • Expected outcome: Consensus functional annotation with confidence scores

  • Validation: Consistency checking across multiple prediction methods

Common bean transcriptome analysis has resulted in a significant increase in available ESTs, providing a platform for functional genomics and improving annotation of uncharacterized proteins . The 768-marker array of single nucleotide polymorphisms based on Trans-legume Orthologous Group (TOG) genes provides additional resources for functional prediction through comparative genomics .

How can I evaluate the immunogenicity of recombinant Phaseolus vulgaris 43 kDa cell wall protein?

Evaluating immunogenicity requires a multi-level experimental approach:

• In silico prediction:

  • Methodology: B-cell epitope prediction using BepiPred and DiscoTope

  • Expected outcome: Identification of potential surface-exposed epitopes

  • Tools: T-cell epitope prediction using NetMHCpan and IEDB analysis resources

• Antibody generation:

  • Methodology: Immunization protocol in rabbits or mice with purified recombinant protein

  • Expected outcome: High-titer polyclonal antibodies

  • Validation: ELISA and Western blot to confirm specificity

• Epitope mapping:

  • Methodology: Overlapping peptide arrays spanning the complete protein sequence

  • Expected outcome: Identification of immunodominant regions

  • Analysis: Correlation with predicted epitopes and structural features

• Cellular immune response:

  • Methodology: T-cell proliferation assays using peripheral blood mononuclear cells

  • Expected outcome: Identification of T-cell stimulatory capacity

  • Cytokine profiling: Measurement of cytokine secretion patterns following stimulation

Sample preparation approaches used in proteomic studies of Phaseolus vulgaris can be adapted for immunological analysis, including tryptic digestion protocols for epitope mapping by mass spectrometry .