Recombinant Oryza sativa subsp. japonica UPF0496 protein 3 (Os03g0148000, LOC_Os03g05440)

What is UPF0496 protein 3 in Oryza sativa subsp. japonica and how is it classified?

UPF0496 protein 3 is a protein encoded by the Os03g0148000 gene (also designated as LOC_Os03g05440) in rice (Oryza sativa subsp. japonica). It belongs to the UPF0496 protein family, where "UPF" stands for "Uncharacterized Protein Family," indicating that its precise function remains to be fully characterized. It is a full-length protein consisting of 378 amino acids and can be recombinantly produced with an N-terminal His-tag using E. coli expression systems .

The protein is one of several UPF0496 family members identified in rice, suggesting potential functional diversity within this protein family. Current research indicates that this protein may be involved in cellular processes unique to rice or other cereal crops, though specific pathways are still being elucidated.

How do gene identifiers Os03g0148000 and LOC_Os03g05440 relate to each other?

Os03g0148000 and LOC_Os03g05440 are different identifiers for the same gene that encodes UPF0496 protein 3. This dual nomenclature stems from different annotation systems used in rice genomics:

Researchers should be aware of both identifiers when conducting literature searches, as publications may use either nomenclature. Both designations refer to the same genomic locus on chromosome 3 of rice, which encodes the UPF0496 protein 3 .

What distinguishes UPF0496 protein 3 from other members of the UPF0496 protein family in rice?

UPF0496 protein 3 is one of several UPF0496 family members identified in rice, with notable differences in amino acid sequence length and composition compared to its homologs:

The variation in sequence length (UPF0496 protein 3 being the shortest at 378 amino acids compared to protein 2 at 408 and protein 4 at 456) suggests potential functional diversification among these proteins . The "putative" designation for UPF0496 protein 2 indicates less certainty about its functional classification. These differences highlight the importance of studying each family member individually to understand their specific roles in rice biology.

What are the optimal conditions for expression and purification of recombinant UPF0496 protein 3?

Based on protocols for similar UPF0496 proteins, the following expression and purification conditions are recommended:

• Expression System: E. coli (typically BL21(DE3) or derivatives)

• Expression Construct: N-terminal His-tagged fusion protein

• Induction Parameters:

  • Temperature: 16-25°C (lower temperatures often improve soluble expression)

  • IPTG concentration: 0.1-1.0 mM

  • Induction duration: 4-16 hours

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) using the His-tag

  • Buffer optimization to maintain protein stability

  • Quality control via SDS-PAGE to ensure >90% purity

After purification, the protein is typically obtained as a lyophilized powder, which should be reconstituted according to specific storage buffer requirements (Tris/PBS-based buffer with 6% Trehalose, pH 8.0) . This approach has been successfully applied to other UPF0496 family members and should be adaptable for UPF0496 protein 3.

How can researchers validate the purity and integrity of recombinant UPF0496 protein 3?

Validation of recombinant UPF0496 protein 3 requires a multi-step approach to ensure both purity and functional integrity:

• SDS-PAGE Analysis:

  • Separate purified protein via SDS-PAGE

  • Stain with Coomassie blue

  • Verify single band at expected molecular weight

  • Confirm purity >90%

• Western Blotting Protocol:

  • Electrotransfer to PVDF membrane at 100V for 60 min

  • Block with 5% non-fat milk in TTBS (0.2M TRIS-HCl pH 7.6, 1.37M NaCl, 0.1% Tween-20)

  • Probe with specific antibodies against either the protein or His-tag

  • Develop with ECL substrate and document

• Mass Spectrometry:

  • Peptide mass fingerprinting to confirm identity

  • Sequence coverage analysis

  • Post-translational modification assessment

• Functional Assays:

  • Protein-protein interaction studies

  • Enzymatic activity tests (if applicable)

This comprehensive validation approach ensures both the physical and functional quality of the recombinant protein preparation .

What methods are most effective for extracting native UPF0496 protein 3 from rice tissues?

For effective extraction of native UPF0496 protein 3 from rice tissues, the following protocol has been optimized:

• Grind rice tissue into a fine powder in liquid nitrogen

• Add 800 μl extraction buffer to each 300 mg powder sample

• Vortex thoroughly and chill on ice for 10 minutes

• Centrifuge at 12,000 rpm for 10 minutes at 4°C

• Collect the supernatant and store at -70°C

Extraction Buffer Composition:

• 62.5 mM TRIS-HCl (pH 7.4)

• 10% glycerol

• 0.1% SDS

• 2 mM EDTA

• 1 mM phenylmethylsulphonyl fluoride (PMSF)

• 5% (v/v) β-mercaptoethanol

Protein concentration can be determined using the Bradford method. This extraction approach has been validated for various rice proteins and should be effective for UPF0496 protein 3 . For tissue-specific studies, researchers should consider developmental stage and growth conditions, as protein expression patterns may vary significantly.

What are the optimal storage conditions for maintaining stability of recombinant UPF0496 protein 3?

Based on established protocols for UPF0496 family proteins, the following storage conditions are recommended to maintain stability and activity:

It is strongly recommended to briefly centrifuge vials prior to opening to bring contents to the bottom. After reconstitution, the addition of glycerol is critical for maintaining long-term stability during frozen storage . These conditions have been validated for maintaining protein integrity and should be strictly followed to ensure experimental reproducibility.

How can solubility issues with recombinant UPF0496 protein 3 be addressed?

Solubility challenges with UPF0496 protein 3 can be addressed through multiple strategies:

• Expression Optimization:

  • Lower induction temperature (16-18°C)

  • Reduce IPTG concentration (0.1-0.2 mM)

  • Co-express with molecular chaperones

  • Consider specialized E. coli strains (e.g., Rosetta, Arctic Express)

• Buffer Optimization:

  • Adjust pH (try range of 7.0-8.5)

  • Modify ionic strength (150-500 mM NaCl)

  • Add solubility enhancers:

    • 6% Trehalose (as used in standard storage buffer)

    • 10% glycerol

    • 0.1% non-ionic detergents for membrane-associated proteins

    • 1-5 mM reducing agents (DTT, β-mercaptoethanol)

• Refolding Strategies:

  • If inclusion bodies form, optimize solubilization and refolding

  • Gradual dialysis to remove denaturants

  • On-column refolding during purification

These approaches should be systematically tested to determine the optimal conditions for maintaining UPF0496 protein 3 in solution . The addition of 6% Trehalose in the storage buffer suggests this additive may be particularly effective for this protein family.

What approaches can be used to determine the biological function of UPF0496 protein 3 in rice?

Elucidating the biological function of UPF0496 protein 3 requires a multi-faceted research approach:

• Gene Expression Analysis:

  • Transcriptome profiling across tissues and developmental stages

  • Response to environmental stresses and hormonal treatments

  • Co-expression network analysis to identify functional associations

• Genetic Modification Studies:

  • CRISPR/Cas9 knockout or knockdown lines

  • Overexpression transgenic plants

  • Phenotypic characterization under various conditions

• Protein Interaction Studies:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation with putative partners

  • Protein complex isolation and characterization

• Subcellular Localization:

  • Fluorescent protein fusions (GFP, YFP)

  • Immunolocalization using specific antibodies

  • Cell fractionation followed by western blotting

• Functional Complementation:

  • Heterologous expression in model organisms

  • Rescue of knockout phenotypes

This systematic approach would help position UPF0496 protein 3 within cellular pathways and biological processes. The in vitro fertilization (IVF) system described for rice zygote development studies could be adapted to investigate potential roles in early development .

How can recombinant UPF0496 protein 3 be used as a research tool in rice molecular biology?

Recombinant UPF0496 protein 3 can serve as a valuable research tool in multiple applications:

• Antibody Generation:

  • Production of polyclonal or monoclonal antibodies

  • Development of immunological assays for native protein detection

• Protein Interaction Studies:

  • In vitro pull-down assays to validate interaction partners

  • Surface plasmon resonance for binding kinetics

  • Protein arrays for interaction screening

• Structural Biology:

  • Crystallization trials for X-ray diffraction studies

  • NMR analysis for solution structure determination

  • Cryo-EM for larger complexes

• Biochemical Assays:

  • Substrate identification

  • Enzymatic activity characterization

  • Post-translational modification analysis

• Quantitative Western Blotting:

  • Standard curve generation using purified recombinant protein

  • Absolute quantification of native protein in rice tissues

These applications leverage the availability of highly purified recombinant protein to advance our understanding of UPF0496 protein 3's role in rice biology . The western blotting standardization approach described in the research literature is particularly valuable for quantitative expression studies.

What comparative genomics approaches can reveal evolutionary insights about UPF0496 protein 3?

Comprehensive evolutionary analysis of UPF0496 protein 3 can provide valuable insights into its conservation and functional importance:

• Phylogenetic Analysis:

  • Sequence alignment of UPF0496 homologs across plant species

  • Construction of phylogenetic trees to visualize evolutionary relationships

  • Identification of clade-specific features

• Selective Pressure Analysis:

  • Calculation of Ka/Ks ratios to identify regions under selection

  • Identification of conserved domains versus variable regions

  • Inference of functional constraints

• Structural Conservation:

  • Homology modeling based on structurally characterized proteins

  • Mapping of conserved residues onto predicted structures

  • Identification of potential functional sites

• Synteny Analysis:

  • Examination of genomic context across species

  • Identification of conserved gene neighborhoods

  • Analysis of duplication patterns within rice subspecies

• Expression Pattern Comparison:

  • Cross-species comparison of expression profiles

  • Correlation with physiological or developmental processes

This evolutionary perspective would help determine whether UPF0496 protein 3 serves a rice-specific function or plays a more conserved role across plant lineages, providing context for functional studies.

How can researchers address antibody cross-reactivity issues when studying UPF0496 protein family members?

Addressing antibody cross-reactivity is crucial when studying closely related proteins like UPF0496 family members:

• Epitope Selection Strategy:

  • Perform sequence alignment of all UPF0496 family proteins

  • Identify unique regions specific to protein 3

  • Select peptide epitopes with minimal homology to other family members

  • Avoid conserved domains shared across the family

• Antibody Production Approach:

  • Use full-length recombinant protein for immunization

  • Alternatively, use synthetic peptides from unique regions

  • Screen antisera against multiple UPF0496 proteins to assess specificity

  • Purify antibodies using affinity chromatography against specific antigen

• Validation Methods:

  • Western blotting against recombinant proteins of all family members

  • Immunoprecipitation followed by mass spectrometry

  • Testing in knockout/knockdown lines as negative controls

  • Preabsorption tests with competing antigens

• Experimental Controls:

  • Include samples from plants lacking the target protein

  • Use recombinant proteins as positive controls

  • Employ epitope-tagged versions in transgenic plants

These strategies minimize cross-reactivity issues and ensure reliable detection of the specific UPF0496 protein 3 in experimental systems .

What are the critical quality control parameters for recombinant UPF0496 protein 3 production?

Quality control is essential for ensuring reliable experimental results with recombinant UPF0496 protein 3:

• Expression Yield Assessment:

  • Quantification of protein concentration

  • Calculation of yield per liter of culture

  • Batch-to-batch consistency evaluation

• Purity Determination:

  • SDS-PAGE with densitometry (target: >90% purity)

  • Absence of significant contaminant bands

  • High-performance liquid chromatography (HPLC) analysis

• Identity Confirmation:

  • Western blotting with anti-His and protein-specific antibodies

  • Mass spectrometry for molecular weight confirmation

  • N-terminal sequencing for first 5-10 amino acids

• Functional Assessment:

  • Binding assays with known interaction partners (if identified)

  • Enzymatic activity tests (if applicable)

  • Proper folding verification via circular dichroism

• Stability Monitoring:

  • Accelerated stability studies at different temperatures

  • Freeze-thaw cycle tolerance

  • Long-term storage stability assessment

These quality control parameters should be documented for each batch of recombinant protein to ensure experimental reproducibility and reliability of research findings .

What advanced analytical techniques can be applied to characterize UPF0496 protein 3?

Several sophisticated analytical techniques can provide detailed characterization of UPF0496 protein 3:

These techniques provide complementary information about the protein's structure, stability, and function, offering deeper insights than routine biochemical assays alone. Integration of multiple approaches provides the most comprehensive characterization of UPF0496 protein 3.