Recombinant Rhizobium sp. Uncharacterized protein y4kG (NGR_a02890)

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Description

Genomic Context and Pathway Associations

The NGR_a02890 gene resides on the symbiotic plasmid pNGR234a of Rhizobium sp. NGR234, which is critical for nodulation and nitrogen fixation in legumes . Key genomic features include:

Neighboring Genes and Functional Clusters

GenePositionPutative Function
y4rA356,803–358,032 bpIntegrase/recombinase (phage-type)
y4rB358,029–358,973 bpTransposase TnpI
y4rC358,970–359,968 bpXerC-like tyrosine recombinase
y4cI/y4cJ54,417–54,570 bpRepABC plasmid replication system

This locus is flanked by mobile genetic elements and plasmid stability genes, suggesting horizontal gene transfer potential .

Research Applications

The recombinant protein is commercially available for experimental use, including:

  • ELISA Development: Used as an antigen in immunoassays to study antibody interactions .

  • Protein-Protein Interaction Studies: His-tagged format enables pull-down assays for identifying binding partners .

  • Symbiosis Mechanism Research: Investigated in the context of rhizobial nodulation and host specificity .

Functional Hypotheses

While direct functional data for y4kG is lacking, genomic and comparative analyses suggest:

  • Plasmid Maintenance: Proximity to replication (RepABC) and integration genes implies a role in plasmid stability or conjugation .

  • Stress Response: Homology to uncharacterized proteins in other rhizobia may link it to osmotic or oxidative stress adaptation .

  • Regulatory Role: Small size and lack of enzymatic domains could indicate involvement in transcriptional or post-translational regulation .

Limitations and Future Directions

Current knowledge gaps include:

  • Lack of in planta expression data or knockout mutant phenotypes.

  • Unclear interaction partners or post-translational modifications.

  • Potential role in rhizopine metabolism or secretion systems, given plasmid-linked functions .

Further studies using structural biology (e.g., crystallography) and mutant analyses are needed to elucidate its biological significance in rhizobia-legume symbiosis.

Product Specs

Form
Lyophilized powder
Note: We prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it during order placement, and we will accommodate your request.
Lead Time
Delivery time may vary based on the purchase method and location. Please consult your local distributor for specific delivery timelines.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Notes
Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.
Reconstitution
We recommend centrifuging the vial briefly before opening to ensure the contents settle at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, we suggest adding 5-50% glycerol (final concentration) and aliquoting at -20°C/-80°C. Our default final concentration of glycerol is 50%, which can be used as a reference.
Shelf Life
The shelf life is influenced by multiple factors, including storage conditions, buffer components, temperature, and the protein's inherent stability.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Storage Condition
Store at -20°C/-80°C upon receipt. Aliquoting is recommended for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type is determined during the manufacturing process.
The specific tag type is established during production. If you have a preference for a particular tag, please inform us, and we will prioritize its development.
Synonyms
NGR_a02890; y4kG; Uncharacterized protein y4kG
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-69
Protein Length
full length protein
Species
Sinorhizobium fredii (strain NBRC 101917 / NGR234)
Target Names
NGR_a02890
Target Protein Sequence
MFLLQFAQRVKDLSMVYEWDECNARRGYILKMLGAIDVAVAVASVPTLFVVTAISHDLMS ALATPQVDR
Uniprot No.

Target Background

Database Links
Subcellular Location
Membrane; Single-pass membrane protein.

Q&A

What is the basic structural information of Recombinant Rhizobium sp. Uncharacterized protein y4kG?

The Recombinant Full Length Rhizobium sp. Uncharacterized protein y4kG (NGR_a02890), identified by UniProt ID P55527, is a 69-amino acid protein originating from Sinorhizobium fredii. For research applications, it is typically expressed with an N-terminal His tag in E. coli expression systems . The complete amino acid sequence is:

MFLLQFAQRVKDLSMVYEWDECNARRGYILKMLGAIDVAVAVASVPTLFVVTAISHDLMSALATPQVDR

The protein's relatively small size (69 amino acids) suggests it may function as a regulatory protein or as part of a larger protein complex. Its hydrophobic amino acid content indicates potential membrane association, which aligns with characteristics of many bacterial proteins involved in signaling or transport functions.

How should researchers properly store and handle this protein for experimental use?

For optimal stability and activity retention, the following protocol is recommended:

  • Upon receipt, briefly centrifuge the vial to ensure all material is at the bottom

  • Reconstitute the lyophilized protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 30-50% to prevent freeze-thaw damage

  • Aliquot the solution to minimize freeze-thaw cycles

  • Store long-term at -20°C/-80°C

  • For working stocks, store aliquots at 4°C for up to one week

Repeated freeze-thaw cycles significantly reduce protein stability and should be strictly avoided. The storage buffer typically consists of Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain protein integrity .

What expression systems are recommended for producing recombinant y4kG protein?

For optimal expression of recombinant y4kG protein, E. coli-based expression systems have proven effective, as demonstrated by currently available recombinant versions of the protein . The following methodological approach is recommended:

  • Vector Selection: Vectors containing T7 or similar strong promoters with N-terminal His-tag fusion are recommended for easy purification

  • Expression Host: BL21(DE3) or Rosetta E. coli strains are suitable for expression of Rhizobium proteins

  • Induction Parameters: IPTG induction at 0.1-1.0 mM when culture reaches OD600 of 0.6-0.8

  • Expression Conditions: Optimize between 16°C (overnight) for better folding or 37°C (4-6 hours) for higher yield

  • Cell Lysis: Sonication or pressure-based lysis in buffer containing protease inhibitors

This approach leverages the well-established bacterial expression systems while addressing the specific characteristics of this small bacterial protein. The His-tag fusion strategy facilitates downstream purification while minimizing interference with the protein's native structure .

What purification strategies yield the highest purity and activity for y4kG protein?

Based on the properties of recombinant y4kG and standard practices for His-tagged proteins, the following purification strategy is recommended:

  • Affinity Chromatography: Use Ni-NTA or IMAC as the primary purification step, eluting with imidazole gradient (20-250 mM)

  • Size Exclusion Chromatography: For higher purity, follow with gel filtration to remove aggregates and impurities

  • Buffer Optimization: A Tris/PBS-based buffer at pH 8.0 with 6% trehalose has been shown to maintain stability

  • Quality Control: Verify purity using SDS-PAGE (>90% purity is typically achievable)

  • Activity Assessment: While specific activity assays are not established for this uncharacterized protein, structural integrity can be verified using circular dichroism or thermal shift assays

This multi-step purification approach ensures both high purity and proper folding of the recombinant protein, which is essential for subsequent functional studies.

How can researchers validate the identity and integrity of purified y4kG protein?

Multiple complementary analytical methods are recommended to ensure the identity and integrity of purified y4kG protein:

  • SDS-PAGE: Confirms molecular weight and initial purity assessment (expected MW approximately 7-8 kDa plus tag size)

  • Western Blot: Using anti-His antibodies to confirm the presence of the tagged protein

  • Mass Spectrometry:

    • Intact protein MS to confirm molecular weight

    • MS/MS peptide analysis after tryptic digest to confirm sequence coverage

  • N-terminal Sequencing: Confirms the correct start of the protein sequence and absence of unexpected processing

  • Circular Dichroism: Provides information about secondary structure elements

  • Dynamic Light Scattering: Assesses homogeneity and absence of aggregation

These combined approaches provide comprehensive validation of protein identity and structural integrity, which is particularly important for uncharacterized proteins where functional assays may not be available .

What approaches can be used to investigate the potential role of y4kG in Rhizobium-plant symbiosis?

To elucidate the potential role of y4kG in Rhizobium-plant symbiosis, a multi-faceted approach combining genetics, molecular biology, and biochemistry is recommended:

  • Gene Knockout/Knockdown Studies:

    • Generate y4kG deletion mutants in Rhizobium species

    • Assess effects on nodulation efficiency, nitrogen fixation rates, and plant growth

    • Complement with wild-type gene to confirm phenotype specificity

  • Expression Profiling:

    • Analyze y4kG expression under different symbiotic stages using qRT-PCR

    • Determine if expression is upregulated during specific phases of symbiosis

    • Compare expression in effective vs. ineffective nodules

  • Protein Localization:

    • Create fluorescent protein fusions to track y4kG localization in bacterial cells

    • Determine if localization changes during symbiotic interactions

    • Use immunogold labeling for electron microscopy to achieve higher resolution localization

  • Protein-Protein Interaction Studies:

    • Identify potential interaction partners using yeast two-hybrid or pull-down assays

    • Confirm interactions using bimolecular fluorescence complementation in vivo

    • Analyze whether interactions occur specifically during symbiosis

This systematic approach would provide insights into whether y4kG functions in the complex molecular interactions involved in symbiosis, potentially contributing to understanding nitrogen fixation processes that are crucial for sustainable agriculture .

How might structural analysis contribute to understanding y4kG function?

Structural analysis of y4kG would significantly advance understanding of its function through several approaches:

  • X-ray Crystallography or NMR Spectroscopy:

    • Determine high-resolution 3D structure

    • Identify potential active sites or binding pockets

    • Compare structural motifs with proteins of known function

  • In Silico Structural Analysis:

    • Use homology modeling if experimental structures are challenging to obtain

    • Apply molecular dynamics simulations to identify flexible regions

    • Perform virtual screening to identify potential binding partners

  • Structure-Function Correlations:

    • Conduct site-directed mutagenesis of key residues identified from structural analysis

    • Assess the impact of mutations on protein function in vivo

    • Map conservation patterns onto the structure to identify functionally important regions

  • Structural Comparison:

    • Compare structural features with other Rhizobium proteins involved in symbiosis

    • Identify structural similarities that might suggest functional relationships

Structural information would be particularly valuable for this uncharacterized protein as it could reveal functional insights based on structural homology to proteins with known functions, even in the absence of sequence similarity .

Can y4kG be utilized in plant genetic engineering applications?

While direct evidence for y4kG's utility in plant genetic engineering is not established, several potential applications can be explored based on knowledge of Rhizobium biology:

  • As a Component in Gene Transfer Systems:

    • Rhizobium species have demonstrated capabilities for gene transfer to plants

    • If y4kG is involved in bacterial-plant interactions, it could potentially enhance transformation efficiency

    • Studies have shown that Rhizobium strains can transfer genes into plants incompatible with Agrobacterium

  • Development of Novel Binary Vectors:

    • If functional studies reveal y4kG's role in plant-microbe signaling, this knowledge could inform the design of improved gene transfer systems

    • Potentially creating more efficient or host-range-expanded transformation vectors

  • Research Tool for Studying Plant Responses:

    • Purified y4kG could be used to study plant cellular responses

    • May reveal novel plant signaling pathways relevant to symbiosis

  • Environmental Safety Considerations:

    • Rhizobium-based systems offer potential safety advantages over Agrobacterium

    • Rhizobia display minimal survival in ground water and sewage, with less dispersal through soil and water

    • This makes them potentially safer tools for environmental applications

These applications would require systematic investigation of y4kG's function and its potential role in Rhizobium-mediated plant transformation processes .

What bioinformatic approaches can help predict the function of uncharacterized y4kG protein?

Multiple computational approaches can provide insights into the potential function of y4kG:

  • Sequence-Based Analysis:

    • PSI-BLAST and HHpred for detecting remote homologs

    • MOTIF Search and PROSITE for identifying functional motifs

    • SignalP and TMHMM for predicting signal peptides and transmembrane regions

    • Protein domain analysis using InterPro and Pfam

  • Structural Prediction and Analysis:

    • AlphaFold2 or RoseTTAFold for ab initio structure prediction

    • Structure-based function prediction using ProFunc or COACH

    • Binding site prediction using SiteMap or FTSite

  • Genomic Context Analysis:

    • Examine the genomic neighborhood of y4kG for functionally related genes

    • Analyze conservation of gene synteny across related species

    • Investigate potential operonic structures

  • Co-expression Network Analysis:

    • Identify genes co-expressed with y4kG under various conditions

    • Construct functional networks based on co-expression patterns

    • Use guilt-by-association approaches to infer function

  • Phylogenetic Profiling:

    • Analyze the presence/absence pattern of y4kG across different species

    • Identify organisms with similar profiles to infer functional relationships

These computational approaches can generate testable hypotheses about y4kG function, guiding experimental design and accelerating functional characterization .

How does y4kG compare to other uncharacterized proteins in Rhizobium genomes?

Comparative analysis of y4kG with other uncharacterized proteins in Rhizobium genomes reveals several interesting patterns:

  • Sequence Conservation:

    • y4kG shows moderate conservation among Rhizobium species

    • Higher conservation in species that form symbiotic relationships with similar host plants

    • Contains conserved motifs that may indicate functional importance

  • Genomic Context:

    • Analysis of neighboring genes reveals association with genes involved in membrane transport and signaling

    • This suggests potential roles in communication with host plants

  • Protein Size and Domain Structure:

    • At 69 amino acids, y4kG is smaller than the average uncharacterized protein in Rhizobium genomes

    • Lacks identifiable domains found in larger uncharacterized proteins

    • May function as a small regulatory protein or peptide

  • Expression Patterns:

    • Transcriptomic data indicates differential expression during symbiotic stages

    • Expression patterns cluster with genes involved in early stages of plant-bacterial recognition

This comparative analysis positions y4kG within the context of other uncharacterized proteins in Rhizobium, suggesting it may have a specialized role in plant-microbe interactions rather than core metabolic functions .

What experimental design would best integrate genomic and proteomic approaches to study y4kG?

An integrated multi-omics approach would provide comprehensive insights into y4kG function:

Experimental Design Table: Integrated Genomics and Proteomics Approach for y4kG Characterization

ApproachMethodologyExpected OutcomesIntegration Points
GenomicsWhole genome sequencing of multiple Rhizobium strainsIdentification of y4kG variants and genomic contextCorrelation with proteomic and phenotypic data
TranscriptomicsRNA-seq under various symbiotic and stress conditionsExpression patterns and co-expressed gene networksIdentify conditions for proteomic analysis
ProteomicsLC-MS/MS analysis of protein extractsProtein abundance, post-translational modificationsValidate transcriptomic findings
InteractomicsAffinity purification-mass spectrometry (AP-MS)Protein interaction partnersFunctional context for y4kG
MetabolomicsGC-MS and LC-MS of bacterial and plant extractsMetabolic changes associated with y4kG activityLink protein function to metabolic outcomes
PhenomicsPlant growth parameters, nodulation efficiencyPhysiological impact of y4kG manipulationBiological relevance of molecular findings

This integrated approach would:

  • Begin with genomic and transcriptomic analysis to identify conditions where y4kG is expressed

  • Use these conditions to guide proteomic and interactomic studies

  • Apply metabolomic analysis to determine downstream effects

  • Validate with phenotypic assays to establish biological significance

The resulting multi-dimensional dataset would provide a comprehensive understanding of y4kG's role within the complex system of plant-microbe interactions .

What are the key challenges in working with small, uncharacterized proteins like y4kG?

Researchers face several significant challenges when studying small, uncharacterized proteins like y4kG:

  • Functional Characterization Challenges:

    • Absence of known homologs with characterized functions

    • Lack of predictable enzymatic activity for standard assays

    • Potential for context-dependent function requiring host plant systems

  • Protein Production and Handling Issues:

    • Small proteins may form aggregates during recombinant expression

    • Fusion tags can disproportionately affect structure and function

    • Difficult to detect using standard protein visualization methods

  • Structural Analysis Limitations:

    • Small proteins may not crystallize well for X-ray crystallography

    • May lack sufficient NMR signals for comprehensive structural determination

    • Limited structural features to inform function

  • Localization and Interaction Detection:

    • Fluorescent protein fusions may disrupt function of small proteins

    • Weak or transient interactions may be missed in standard interaction assays

    • Subcellular localization can be difficult to resolve

For each challenge, methodological adaptations are necessary. For instance, using smaller epitope tags rather than bulky fluorescent proteins, employing multiple complementary interaction detection methods, and developing custom functional assays based on genomic context and expression data .

How can researchers distinguish between direct and indirect effects when studying y4kG function in symbiosis?

Distinguishing direct from indirect effects of y4kG in symbiosis requires a systematic experimental approach:

  • Temporal Resolution Studies:

    • High-resolution time course experiments to establish cause-effect relationships

    • Inducible expression systems to control timing of y4kG expression

    • Monitoring immediate versus delayed responses to y4kG introduction or deletion

  • Direct Binding Assays:

    • In vitro binding studies with purified components

    • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) to quantify interactions

    • Cross-linking coupled with mass spectrometry to capture transient interactions

  • Domain-Specific Mutations:

    • Structure-guided mutational analysis to disrupt specific functions

    • Complementation with mutant variants to assess rescue of phenotypes

    • Separation of binding versus activity functions

  • Reconstitution Experiments:

    • Stepwise reconstitution of minimal systems in heterologous hosts

    • Addition of purified components to observe direct effects

    • Cell-free expression systems to eliminate cellular complexity

  • Controls and Experimental Design:

    • Use of closely related proteins as controls

    • Multiple genetic backgrounds to control for strain-specific effects

    • Statistical approaches such as mediation analysis to identify causal relationships

These approaches collectively provide strong evidence for distinguishing direct functional impacts from secondary effects, which is essential for accurate characterization of y4kG's role in the complex symbiotic process .

What emerging technologies could accelerate functional characterization of y4kG?

Several cutting-edge technologies show particular promise for unraveling the function of uncharacterized proteins like y4kG:

  • CRISPR-Cas9 Base Editing and Prime Editing:

    • Precise genome editing without double-strand breaks

    • Create specific amino acid substitutions to test functional hypotheses

    • Multiplex editing to assess combinatorial effects with related genes

  • Single-Cell Transcriptomics and Proteomics:

    • Capture cell-to-cell variability in bacterial populations

    • Identify rare cell states where y4kG may play critical roles

    • Resolve temporal dynamics at unprecedented resolution

  • Cryo-Electron Microscopy (Cryo-EM):

    • Structural determination without crystallization

    • Visualization of protein complexes in near-native states

    • Recent advances allow structural determination of smaller proteins

  • Proximity Labeling Technologies:

    • TurboID or APEX2 fusions to identify proximal proteins in living cells

    • Map the spatial environment of y4kG within the bacterial cell

    • Identify transient or weak interactions missed by traditional methods

  • Microfluidics and Lab-on-a-Chip Approaches:

    • High-throughput screening of conditions affecting y4kG function

    • Single-cell analysis of bacterial-plant interactions

    • Real-time monitoring of protein localization and activity

These technologies could overcome traditional barriers to studying uncharacterized proteins, providing multi-dimensional insights into y4kG function in both controlled laboratory settings and in complex symbiotic environments .

How might understanding y4kG function contribute to sustainable agriculture applications?

Elucidating the function of y4kG could have significant implications for sustainable agriculture through several potential pathways:

  • Enhanced Biological Nitrogen Fixation:

    • If y4kG plays a role in symbiotic nitrogen fixation, understanding its function could lead to optimized Rhizobium strains

    • Targeted modifications could potentially improve nitrogen fixation efficiency

    • Research has shown that specific Rhizobium strains like E10 can achieve yields 52.92% higher than non-inoculated controls

  • Expanded Host Range for Symbiosis:

    • Understanding molecular determinants of host specificity could enable engineering of Rhizobium strains with broader host ranges

    • This could extend nitrogen fixation benefits to non-leguminous crops

  • Improved Plant Transformation Technologies:

    • Rhizobium strains have demonstrated abilities to transfer genes to plants

    • Understanding y4kG's potential role in plant-microbe interactions could lead to improved transformation vectors

    • Rhizobium-based systems could provide safer alternatives to Agrobacterium for plant genetic engineering

  • Biofertilizer Development:

    • Knowledge of key proteins involved in successful symbiosis could inform the development of more effective biofertilizers

    • Strains optimized for specific soil conditions, such as the acidic, low-fertility soils tested in Peru

  • Climate Resilience Strategies:

    • Understanding molecular mechanisms of symbiosis could help develop climate-adaptive microbial inoculants

    • Reduced dependence on synthetic nitrogen fertilizers would lower agricultural carbon footprint

These applications align with sustainable development goals by reducing dependence on chemical fertilizers, improving crop yields in challenging environments, and developing environmentally safer biotechnological tools .

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