Recombinant Vibrio vulnificus UPF0345 protein VVA0047 (VVA0047)

What is the basic structural characterization of Recombinant Vibrio vulnificus UPF0345 protein VVA0047?

While specific structural data for VVA0047 is limited, research approaches can be modeled after related Vibrio vulnificus membrane proteins. Based on similar proteins like UPF0397 protein VVA0348, researchers should analyze amino acid sequence using bioinformatics tools to predict secondary structure elements. The VVA0348 protein consists of 182 amino acids with specific transmembrane domains that can serve as a comparative model for structural studies of VVA0047 . Methodologically, circular dichroism spectroscopy combined with structural prediction algorithms provides initial characterization, followed by crystallography studies for detailed structure determination.

How does Vibrio vulnificus UPF0345 compare with other bacterial UPF proteins?

UPF (Uncharacterized Protein Family) proteins like VVA0047 require comparative genomic analysis against other bacterial UPF proteins to determine evolutionary relationships and functional conservation. Researchers should employ multiple sequence alignment tools to identify conserved domains across bacterial species, particularly focusing on comparisons with related Vibrio proteins. When examining proteins from pathogenic bacteria like Vibrio vulnificus, it's essential to consider their potential roles in virulence mechanisms, as seen with other Vibrio proteins such as VvhA, VvpE, and RtxA1 .

What are the predicted functional domains of VVA0047 and their implications for research?

Domain prediction for VVA0047 should be approached through bioinformatics tools like InterPro, SMART, and Pfam. Based on analysis of related Vibrio proteins, researchers should examine potential transmembrane regions and functional motifs. For experimental validation of predicted domains, targeted mutagenesis methods can be employed, creating truncated versions of the protein to assess functional changes. As observed with other Vibrio vulnificus proteins, domain function may relate to membrane interactions, potentially contributing to bacterial survival mechanisms or host interaction processes .

What expression systems are optimal for recombinant production of VVA0047?

For recombinant expression of Vibrio vulnificus proteins like VVA0047, E. coli expression systems have proven effective, as demonstrated with the UPF0397 protein VVA0348 . Researchers should consider these methodological approaches:

For membrane-associated proteins, inclusion of solubilizing tags (such as His-tag as used with VVA0348) significantly improves purification efficiency . Expression conditions should be systematically optimized through factorial design experiments varying temperature, inducer concentration, and harvest timing.

What purification strategies yield highest purity and functional integrity for VVA0047?

Based on successful approaches with similar proteins, a multi-step purification strategy is recommended. For His-tagged constructs like those used with VVA0348, immobilized metal affinity chromatography (IMAC) provides an effective initial capture step . This should be followed by secondary purification methods:

• Size exclusion chromatography to separate monomeric from aggregated forms

• Ion exchange chromatography for charge variant separation

• Affinity chromatography with specific ligands if applicable

Critical quality assessment should include SDS-PAGE analysis (targeting >90% purity as achieved with VVA0348), western blotting, and mass spectrometry for molecular weight verification . Functional integrity assessment through activity assays specific to predicted protein function is essential before experimental use.

How can researchers overcome common expression challenges with Vibrio vulnificus recombinant proteins?

Expression of Vibrio proteins often faces challenges including inclusion body formation, improper folding, and low yield. Methodological approaches to address these include:

• Fusion with solubility-enhancing partners (MBP, SUMO, or thioredoxin)

• Co-expression with molecular chaperones (GroEL/GroES system)

• Expression as truncated constructs removing problematic regions

• Screening multiple construct designs with varying N- and C-terminal boundaries

For membrane-associated proteins like many UPF family members, inclusion of appropriate detergents during extraction and purification is critical. Testing a panel of detergents (CHAPS, DDM, or OG) at varying concentrations can identify optimal solubilization conditions while maintaining native structure .

What are the optimal storage conditions for maintaining VVA0047 stability?

For long-term storage of recombinant Vibrio proteins like VVA0047, research with similar proteins such as VVA0348 suggests multiple stability factors require consideration. Based on established protocols for VVA0348, lyophilized protein preparations demonstrate superior long-term stability when stored at -20°C to -80°C . For working solutions, researchers should implement these evidence-based practices:

• Store small working aliquots (50-100 μL) to avoid repeated freeze-thaw cycles, which significantly reduce protein activity

• Maintain short-term working stocks (up to one week) at 4°C as recommended for VVA0348

• Include cryoprotectants like glycerol (5-50%) to prevent freeze-thaw damage, with 50% being optimal for many Vibrio proteins

• Consider protein-specific buffer optimization through thermal shift assays to identify stabilizing additives

Researchers should systematically evaluate stability using activity assays or structural integrity measurements at defined time points to establish protein-specific shelf-life parameters.

How can researchers assess and maintain the functional integrity of VVA0047 during experimental procedures?

Functional integrity assessment requires both structural and activity-based approaches. For structural evaluation, researchers should employ:

• Circular dichroism spectroscopy to monitor secondary structure changes

• Intrinsic tryptophan fluorescence to detect tertiary structure alterations

• Size exclusion chromatography to identify aggregation

For activity assessment, while specific VVA0047 functions remain to be fully characterized, general approaches include:

• Binding assays with predicted interaction partners

• Membrane association studies if predicted to be membrane-localized

• Comparative functional tests with homologous proteins of known function

When handling the protein, maintain temperature control throughout experimental procedures, as thermal stress significantly impacts structural integrity. The addition of stabilizing agents like trehalose (6% as used with VVA0348) can maintain conformational stability during experimental manipulations .

How might VVA0047 contribute to Vibrio vulnificus virulence mechanisms?

Understanding potential virulence contributions requires contextualizing VVA0047 within the broader landscape of Vibrio vulnificus pathogenicity. Unlike well-characterized virulence factors such as VvhA (hemolysin), VvpE (protease), and RtxA1 (cytotoxin) , the specific role of UPF proteins remains less defined. Researchers investigating VVA0047's potential virulence contributions should consider these methodological approaches:

• Generate isogenic knockout mutants to assess changes in virulence phenotypes, similar to approaches used with other Vibrio vulnificus virulence factors

• Examine protein expression levels under infection-relevant conditions (iron limitation, host cell contact)

• Assess potential interactions with known virulence regulatory systems such as the HlyU regulator that controls RtxA1 expression

• Investigate localization patterns during infection processes using immunofluorescence or reporter fusions

While VvhA and VvpE single mutations did not significantly affect cytotoxicity or lethality in infection models , combinatorial approaches examining multiple factors simultaneously may reveal synergistic relationships with UPF proteins like VVA0047.

What experimental models are appropriate for studying VVA0047 in the context of host-pathogen interactions?

Selection of appropriate experimental models should reflect the pathophysiology of Vibrio vulnificus infections. Based on established research approaches with this pathogen, researchers should consider:

When designing host cell interaction studies, researchers should examine the effects of VVA0047 on cellular processes disrupted by other Vibrio proteins, such as cytoskeletal rearrangements, tight junction integrity, and cell death pathways observed with RtxA1 toxin . The requirement for cell contact observed with some Vibrio virulence mechanisms suggests examining VVA0047 in direct co-culture systems rather than using filtered supernatants alone.

How can structure-function relationship studies of VVA0047 inform therapeutic development against Vibrio vulnificus?

Advanced structure-function studies of VVA0047 require integrated approaches combining structural biology, biophysical characterization, and functional assessment. Researchers should:

• Determine high-resolution structures through X-ray crystallography or cryo-electron microscopy

• Identify functional motifs through systematic alanine scanning mutagenesis

• Characterize protein-protein or protein-ligand interactions through techniques like surface plasmon resonance or isothermal titration calorimetry

• Develop specific inhibitors through structure-based drug design approaches

The potential therapeutic relevance derives from Vibrio vulnificus's severe clinical manifestations, including septicemia with mortality rates exceeding 50% . If VVA0047 contributes to virulence or essential cellular functions, inhibitor development targeting conserved functional domains could provide novel therapeutic strategies against this highly lethal pathogen.

What are the methodological approaches for examining VVA0047 regulation during environmental stress and host adaptation?

Investigating regulatory mechanisms governing VVA0047 expression requires multi-level analysis of transcriptional, translational, and post-translational control. Advanced methodological approaches include:

• Transcriptional regulation:

  • Promoter mapping and reporter fusion assays

  • ChIP-seq to identify transcription factor binding sites

  • RNA-seq under varying environmental conditions (temperature, pH, osmolarity)

• Environmental response characterization:

  • qRT-PCR analysis of gene expression under infection-relevant conditions

  • Examination of iron-responsive regulation (critical for many Vibrio virulence factors)

  • Assessment of host cell contact-dependent expression changes

• Post-translational regulation:

  • Phosphoproteomic analysis to identify modification sites

  • Protein stability studies under varying environmental conditions

  • Protein-protein interaction network mapping using pull-down assays coupled with mass spectrometry

These approaches should be integrated with comparative analysis across Vibrio species to identify conserved regulatory mechanisms governing UPF protein expression in pathogenic contexts.

How can researchers leverage comparative genomics to better understand VVA0047 evolution and functional conservation across Vibrio species?

Comparative genomic analysis of VVA0047 homologs provides critical evolutionary context for functional studies. Advanced research approaches include:

• Phylogenetic analysis across the Vibrionaceae family to trace evolutionary history

• Identification of selection pressures through dN/dS ratio analysis across coding sequences

• Examination of genomic context conservation to identify potential operonic structures

• Analysis of horizontal gene transfer events that may have influenced protein distribution

Functional predictions derived from evolutionary analysis should be experimentally validated through heterologous expression and complementation studies in multiple Vibrio species. Researchers should examine whether VVA0047 homologs complement functional deficiencies across species boundaries, providing insight into fundamental versus species-specific roles.