Recombinant UPF0283 membrane protein VP1870 (VP1870)

What is UPF0283 membrane protein VP1870 and what organism does it originate from?

UPF0283 membrane protein VP1870 (UniProt ID: Q87NJ8) is a full-length (346 amino acid) membrane protein derived from Vibrio parahaemolyticus Serotype O3:K6. It belongs to the UPF0283 protein family, which consists of proteins with currently unknown functions. The protein's membrane localization suggests potential roles in cellular transport, signaling, or structural functions within bacterial cells. The "UPF" designation (Uncharacterized Protein Family) indicates that while the protein has been identified and sequenced, its precise biological function remains to be fully elucidated through ongoing research efforts .

What expression systems can be used to produce recombinant VP1870 protein?

Recombinant UPF0283 membrane protein VP1870 can be expressed and purified from multiple host systems, each offering distinct advantages. Escherichia coli and yeast expression systems provide the highest protein yields and shorter production timeframes, making them ideal for initial characterization studies or when larger quantities are required. For applications requiring proper protein folding and post-translational modifications, expression in insect cells via baculovirus vectors or in mammalian cell systems is recommended, although these typically result in lower yields and longer production times . The choice of expression system should be determined by the specific requirements of your experimental design and downstream applications.

What are the recommended storage conditions for recombinant VP1870 protein?

For optimal stability and preservation of biological activity, recombinant VP1870 protein should be stored at -20°C to -80°C upon receipt. The protein requires proper aliquoting for multiple use scenarios to minimize freeze-thaw cycles, which can compromise protein integrity and functionality. For short-term usage within one week, working aliquots can be stored at 4°C. The protein is typically supplied in a stabilized Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which helps maintain protein stability during storage . These storage recommendations are crucial for maintaining protein quality throughout the research timeline and ensuring consistent experimental results.

What reconstitution protocol is recommended for lyophilized VP1870 protein?

The optimal reconstitution protocol for lyophilized VP1870 protein involves several critical steps:

• First, briefly centrifuge the vial to bring all contents to the bottom before opening.

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

• Add glycerol to a final concentration between 5-50% (with 50% being standard) to enhance protein stability.

• Properly aliquot the reconstituted protein to minimize freeze-thaw cycles.

• Store aliquots at -20°C/-80°C for long-term preservation of activity .

This methodical approach ensures maximum recovery of active protein and minimizes potential degradation during the reconstitution process.

How should researchers design experiments to evaluate VP1870 protein function?

When designing experiments to evaluate VP1870 protein function, researchers should implement a structured experimental design that:

For membrane proteins like VP1870, functional studies might include membrane localization assays, protein-protein interaction studies, or comparative analyses across different Vibrio species to elucidate evolutionary conservation and functional significance.

What purification methods are most effective for His-tagged VP1870 protein?

For His-tagged VP1870 protein, immobilized metal affinity chromatography (IMAC) represents the primary purification technique due to the N-terminal His-tag fusion. An effective purification protocol typically involves:

• Cell lysis under conditions that preserve protein structure (particularly important for membrane proteins).

• Initial capture using Ni-NTA or similar affinity resin with binding buffer containing low imidazole concentrations (10-20 mM).

• Washing steps with gradually increasing imidazole concentrations to remove non-specifically bound proteins.

• Elution with higher imidazole concentrations (250-500 mM).

• Buffer exchange to remove imidazole using dialysis or size-exclusion chromatography.

• Quality assessment via SDS-PAGE to verify purity (>90% purity is reported for commercial preparations) .

For membrane proteins like VP1870, inclusion of appropriate detergents throughout the purification process is crucial to maintain protein solubility and native conformation.

How does the expression system choice affect post-translational modifications of VP1870?

The choice of expression system significantly impacts post-translational modifications (PTMs) of VP1870, with important implications for protein function and experimental outcomes:

When studying VP1870, researchers should select the expression system based on whether native bacterial PTMs are sufficient or if the experimental design requires specific modifications that might occur in more complex systems . This decision is particularly important for functional studies where protein activity may depend on specific modifications.

What techniques can be employed to study VP1870's membrane topology and structural characteristics?

To investigate VP1870's membrane topology and structural characteristics, researchers can employ multiple complementary approaches:

• Computational prediction tools: Using algorithms like TMHMM, HMMTOP, or Phobius to predict transmembrane regions and topology.

• Biochemical mapping techniques:

  • Cysteine scanning mutagenesis followed by accessibility assays

  • Protease protection assays to identify exposed regions

  • Glycosylation mapping using engineered glycosylation sites

• Structural biology approaches:

  • X-ray crystallography (challenging for membrane proteins, may require lipidic cubic phase)

  • Cryo-electron microscopy (particularly useful for membrane proteins)

  • NMR spectroscopy for dynamic structural information

  • Small-angle X-ray scattering (SAXS) for low-resolution structural information

• Fluorescence-based techniques:

  • FRET (Förster Resonance Energy Transfer) to measure distances between domains

  • GFP-fusion reporter systems to track cellular localization

The purity level of commercially available VP1870 (>90%) makes it suitable for many of these analytical techniques, though additional purification steps may be necessary for high-resolution structural studies.

What considerations should researchers take into account when designing experiments to identify potential interaction partners of VP1870?

When designing experiments to identify VP1870 interaction partners, researchers should consider a multi-faceted approach:

• Experimental design principles:

  • Clearly define the hypothesis regarding potential interaction partners

  • Incorporate appropriate controls to distinguish specific from non-specific interactions

  • Ensure statistical power to detect significant interactions

  • Plan for validation of primary findings through orthogonal methods

• Recommended techniques:

  • Pull-down assays leveraging the His-tag fusion

  • Co-immunoprecipitation with antibodies against VP1870

  • Yeast two-hybrid screening (for soluble domains)

  • Bacterial two-hybrid systems (more suitable for membrane proteins)

  • Proximity labeling approaches (BioID or APEX)

  • Cross-linking mass spectrometry

  • Surface plasmon resonance for quantitative binding studies

• Validation strategies:

  • Reverse pull-down experiments

  • Co-localization studies in bacterial cells

  • Functional assays to demonstrate biological relevance of interactions

  • Mutagenesis to identify critical interaction interfaces

The His-tagged nature of recombinant VP1870 provides an immediate advantage for affinity-based methods, enabling specific capture of the protein and its interacting partners.

What are common challenges in working with recombinant VP1870 and how can they be addressed?

Researchers working with recombinant VP1870 may encounter several challenges, with corresponding solutions:

Adhering to proper storage conditions (-20°C/-80°C) and handling procedures is crucial for maintaining protein integrity and experimental reproducibility . Additionally, working aliquots should be stored at 4°C for no longer than one week to prevent activity loss.

How can researchers verify the quality and activity of VP1870 protein preparations?

To ensure high-quality VP1870 protein preparations for reliable experimental outcomes, researchers should implement a comprehensive quality control protocol:

• Purity assessment:

  • SDS-PAGE analysis with Coomassie staining (should show >90% purity)

  • Western blotting using anti-His antibodies to confirm identity

  • Mass spectrometry to verify protein mass and integrity

• Structural integrity:

  • Circular dichroism spectroscopy to assess secondary structure

  • Thermal shift assays to evaluate protein stability

  • Dynamic light scattering to check for aggregation

• Functional validation:

  • Binding assays with known ligands if available

  • Membrane incorporation studies using liposomes

  • Activity assays based on predicted function (may require development)

• Storage stability monitoring:

  • Comparative analysis of fresh vs. stored samples

  • Regular testing of stored aliquots at defined time points

  • Documentation of protein behavior under various storage conditions

Implementing these quality control measures helps ensure that experimental variations reflect true biological phenomena rather than differences in protein preparation quality.

What are the current knowledge gaps in VP1870 research that warrant further investigation?

Despite available structural and expression information, several knowledge gaps remain in VP1870 research that merit scientific investigation:

• Functional characterization: As a member of the UPF0283 family, the specific biological function of VP1870 remains largely unknown, presenting opportunities for functional genomics approaches.

• Structural determinants: While amino acid sequence data is available , high-resolution structural information is lacking, limiting our understanding of structure-function relationships.

• Interaction network: The protein-protein interaction landscape of VP1870 within Vibrio parahaemolyticus remains unexplored, representing a significant knowledge gap.

• Role in bacterial physiology: How VP1870's membrane localization contributes to cellular processes and bacterial survival requires elucidation.

• Evolutionary conservation: Comparative analysis across Vibrio species and related bacteria could provide insights into functional importance and specialization.

These knowledge gaps present promising avenues for researchers to contribute meaningful advances to the understanding of this membrane protein's biological significance.

What emerging technologies might enhance future studies of VP1870?

Several cutting-edge technologies and approaches hold promise for advancing VP1870 research:

• Structural biology innovations:

  • Advances in cryo-electron microscopy for membrane protein structure determination

  • Integrative structural biology combining multiple data sources

  • AlphaFold2 and other AI-based structure prediction methods

• Functional genomics approaches:

  • CRISPR interference/activation for functional studies in bacterial systems

  • High-throughput phenotypic screening using bacterial mutant libraries

  • Transposon sequencing (Tn-Seq) to identify genetic interactions

• Single-cell technologies:

  • Single-cell transcriptomics to understand expression patterns

  • Super-resolution microscopy for precise localization studies

  • FRET-based biosensors to monitor protein dynamics

• Systems biology integration:

  • Multi-omics data integration to place VP1870 in biological context

  • Network analysis to identify functional modules

  • Computational modeling of membrane protein dynamics

These emerging technologies promise to overcome current technical limitations and provide deeper insights into the structure, function, and biological significance of VP1870, potentially revealing unexpected roles in bacterial physiology or pathogenesis.