Recombinant Shigella boydii serotype 18 UPF0266 membrane protein yobD (yobD)

What is the structural composition of the UPF0266 membrane protein yobD in Shigella boydii serotype 18?

The UPF0266 membrane protein yobD in Shigella boydii serotype 18 (strain CDC 3083-94 / BS512) is a 152-amino acid protein with a predominantly hydrophobic composition. The complete amino acid sequence is: MTITDLVLILFIAALLAFAIYDQFIMPRRNGPTLLAIPLLRRGRIDSVIFVGLIVILIYN NVTNHGALITTWLLSALALMGFYIFWIRVPKIIFKQKGFFFANVWIEYSLIKAMNLSEDG VLVMQLEQRRLLIRVRNIDNLEKIYKLIVSTQ . Analysis of this sequence reveals multiple transmembrane domains consistent with its classification as a membrane protein. The protein contains regions of highly hydrophobic amino acids interspersed with charged residues that likely anchor it within the bacterial membrane, with some portions exposed to either the cytoplasmic or extracellular environment.

How conserved is the yobD protein across different Shigella species?

The yobD protein demonstrates significant conservation across Shigella species, as evidenced by comparative genomic analyses. The UPF0266 membrane protein yobD is present in both Shigella boydii serotype 18 and Shigella sonnei, with highly similar structural and functional characteristics . This conservation suggests evolutionary importance and potential functional significance in Shigella pathogenesis. The high degree of conservation makes yobD a candidate protein for broad-spectrum studies across Shigella species, potentially contributing to its utility in cross-species immunological and functional analyses.

What expression systems are most effective for producing recombinant yobD protein?

E. coli expression systems have been successfully employed for the production of recombinant yobD protein from both Shigella boydii and Shigella sonnei . For optimal expression, researchers should consider the following methodological approach:

• Vector selection: Vectors containing strong inducible promoters (T7, tac) are recommended

• Host strain: E. coli BL21(DE3) or similar strains with reduced protease activity

• Induction conditions: Typically 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Growth temperature: Post-induction temperature reduction to 18-25°C can improve proper folding

• Fusion tags: N-terminal His-tags have been successfully implemented and facilitate purification

The membrane-associated nature of yobD presents challenges for expression and purification, often requiring optimization of detergent conditions during extraction and purification steps to maintain protein stability and native conformation.

What purification strategy yields the highest purity of recombinant yobD protein?

A multi-step purification approach is recommended for obtaining high-purity recombinant yobD protein:

When working with membrane proteins like yobD, inclusion of appropriate detergents (e.g., n-dodecyl-β-D-maltoside or CHAPS) throughout the purification process is crucial to maintain protein solubility and native conformation. The purified protein should be stored at -20°C or -80°C with 50% glycerol to maintain stability for extended periods .

How can researchers validate the structural integrity of purified recombinant yobD protein?

Validation of structural integrity is essential before proceeding with functional studies. A comprehensive validation approach should include:

• SDS-PAGE and Western blotting: Confirm protein size (approximately 17 kDa plus tag size) and immunoreactivity

• Circular dichroism (CD) spectroscopy: Assess secondary structure elements expected in membrane proteins

• Limited proteolysis: Compare digestion patterns with predictions based on sequence

• Thermal shift assays: Evaluate protein stability under various buffer conditions

• Dynamic light scattering (DLS): Determine homogeneity and detect aggregation

• Mass spectrometry: Confirm protein identity and post-translational modifications

For membrane proteins like yobD, additional validation may include reconstitution into liposomes or nanodiscs followed by functional assays to verify proper folding and orientation.

How can recombinant yobD protein be utilized in immunological studies of Shigella infection?

Recombinant yobD protein serves as a valuable tool in immunological studies of Shigella infection through several approaches:

• Antibody production: Purified recombinant yobD can be used to generate polyclonal or monoclonal antibodies for immunolocalization studies, Western blotting, and immunoprecipitation experiments.

• ELISA development: The protein can be employed in enzyme-linked immunosorbent assays to detect anti-yobD antibodies in patient sera, providing insights into immune responses during Shigella infection . This application requires careful optimization of coating conditions to maintain the native epitopes of this membrane protein.

• Proteome microarray incorporation: As demonstrated with other Shigella proteins, yobD can be included in proteome microarrays to profile antibody responses following vaccination or natural infection . This approach allows for high-throughput analysis of immune responses across multiple antigens simultaneously.

• T-cell response analysis: Recombinant yobD can be used in T-cell stimulation assays to assess cell-mediated immune responses, including cytokine production and proliferation assays.

• Cross-reactivity studies: Given the conservation of yobD across Shigella species, the recombinant protein can be used to investigate cross-reactive immune responses that might contribute to broad-spectrum protection .

What is the significance of yobD protein in the context of Shigella proteome microarray studies?

While the yobD protein specifically was not highlighted among the top immunoreactive antigens in the Shigella proteome microarray studies, the microarray platform approach provides important context for understanding potential roles of conserved membrane proteins like yobD . The Shigella proteome microarray containing over 2,000 conserved proteins has demonstrated utility in:

• Identifying immunodominant antigens following vaccination or natural infection

• Comparing immune responses between different vaccine formulations (killed whole-cell vs. live attenuated)

• Correlating antibody profiles with protection against clinical disease

• Identifying novel vaccine targets beyond traditional candidates

The most immunoreactive proteins identified in these studies were components of the type three secretion system (T3SS), including IpaA, IpaB, IpaC, and IpaD . Although yobD was not among these top candidates, its conservation across Shigella species suggests it could still have relevance as part of a broader protein signature or in combination with other antigens.

What potential does yobD protein hold for vaccine development against Shigella infections?

The potential of yobD protein for vaccine development should be considered in the context of broader Shigella vaccine research:

• Conservation advantage: The presence of yobD across Shigella species (S. boydii and S. sonnei demonstrated in the search results ) suggests it could potentially contribute to cross-species protection, addressing the challenge of serotype-specific immunity that limits LPS-based vaccine approaches .

• Microarray context: The Shigella proteome microarray studies have identified a signature of three conserved proteins (IpaA, IpaB, and IpaC) that was predictive of protection against shigellosis . While yobD was not specifically mentioned among these top candidates, the microarray approach demonstrates the value of investigating conserved proteins.

• Complementary antigen: Even if not immunodominant on its own, yobD could potentially serve as a complementary antigen in multi-component vaccine formulations, possibly enhancing broader protection when combined with more immunogenic T3SS components.

• Carrier protein potential: As a membrane protein, yobD could potentially serve as a carrier for conjugate vaccines, presenting Shigella-specific epitopes in an appropriate context.

A systematic approach to evaluating yobD's vaccine potential would include:

• Immunogenicity studies in animal models

• Epitope mapping to identify protective B and T cell epitopes

• Formulation studies with adjuvants appropriate for membrane proteins

• Challenge studies to assess protection against multiple Shigella serotypes

What are the key challenges in structural studies of membrane proteins like yobD?

Structural characterization of membrane proteins like yobD presents several methodological challenges that researchers should anticipate:

• Protein expression: Membrane proteins often express poorly in heterologous systems and may form inclusion bodies. Optimization strategies include:

  • Using specialized E. coli strains (C41, C43) designed for membrane protein expression

  • Employing fusion partners that enhance solubility

  • Testing various induction conditions (temperature, inducer concentration, duration)

• Purification complexity: Maintaining protein stability during extraction from membranes requires careful detergent selection:

  • Initial screening of multiple detergents (DDM, LDAO, CHAPS)

  • Detergent exchange during purification if necessary

  • Consideration of alternative solubilization systems (nanodiscs, amphipols)

• Crystallization difficulties: For X-ray crystallography studies, membrane proteins are notoriously difficult to crystallize:

  • Lipidic cubic phase methods may improve success rates

  • In meso crystallization approaches

  • Antibody fragment co-crystallization to provide crystal contacts

• NMR spectroscopy challenges:

  • Size limitations (yobD at 152 aa may be amenable)

  • Detergent micelle effects on tumbling rates

  • Need for extensive isotopic labeling (15N, 13C, 2H)

• Cryo-EM considerations:

  • Small size of yobD may make it unsuitable as an individual target

  • Complex formation with interaction partners could make it amenable to this technique

How can researchers effectively study protein-protein interactions involving yobD?

Investigating protein-protein interactions for membrane proteins requires specialized approaches:

• Membrane-based yeast two-hybrid systems:

  • Modified yeast two-hybrid systems designed specifically for membrane proteins

  • Split-ubiquitin or MYTH (membrane yeast two-hybrid) systems

• Co-immunoprecipitation optimization:

  • Gentle solubilization conditions to maintain interactions

  • Crosslinking prior to extraction to stabilize transient interactions

  • Mass spectrometry analysis of co-purified proteins

• Proximity labeling approaches:

  • BioID or APEX2 fusion to yobD for in vivo biotinylation of proximal proteins

  • TurboID for rapid labeling in bacterial systems

• Surface plasmon resonance (SPR) studies:

  • Immobilization strategies that maintain native orientation

  • Reconstitution into nanodiscs or liposomes for capture on sensor chips

  • Careful buffer optimization to maintain protein stability

• Fluorescence-based techniques:

  • FRET or BRET studies with candidate interaction partners

  • Fluorescence correlation spectroscopy to study interactions in membranes

• Advanced proteomics approaches:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Chemical crosslinking coupled with mass spectrometry

  • Targeted proteomics to quantify interactions under different conditions