Recombinant Brucella melitensis biotype 1 Immunogenic membrane protein YajC (yajC)

What is the YajC protein in Brucella melitensis and what is its genetic organization?

YajC is a membrane protein encoded by the yajC gene in Brucella species, including B. melitensis and B. abortus. The yajC gene is organized in an operon with the secD gene, both likely involved in protein secretion processes . Nucleotide sequence analysis of a B. abortus 2308 gene library clone (designated MCB68) revealed two open reading frames (ORFs) with significant similarities to YajC and SecD proteins found in several bacterial species . This genetic organization appears to be conserved across Brucella species, which share approximately 99% identity at the DNA level, including B. abortus, B. melitensis, and B. suis .

What immune responses does recombinant YajC protein stimulate when used as an immunogen?

Recombinant YajC protein stimulates both humoral (antibody-mediated) and cell-mediated immune responses. In experimental models:

• Antibody response: Mice inoculated with live B. abortus strains 19 and 2308, as well as B. melitensis RM1, produced antibodies that recognized the recombinant YajC protein in Western blot analyses .

• Cell-mediated response: Splenocytes from mice vaccinated with B. abortus RB51 showed significant proliferation when stimulated in vitro with recombinant maltose binding protein (MBP)-YajC fusion protein. This proliferation was more prominent in 3-day cultures compared to 5-day cultures .

• Cytokine production: The stimulated splenocytes produced gamma interferon (IFN-γ) but not interleukin-4 (IL-4), indicating a predominant T helper 1 (Th1) type immune response , which is associated with protective immunity against intracellular pathogens like Brucella.

The following table shows the concentrations of IFN-γ produced by stimulated splenocytes from vaccinated and naive mice:

• Detectable levels of IFN-γ produced

Why is YajC considered significant for Brucella vaccine development?

YajC is considered significant for Brucella vaccine development for several reasons:

• Immunogenicity: YajC stimulates both humoral and cell-mediated immune responses, particularly a Th1-type response characterized by IFN-γ production, which is critical for protection against intracellular pathogens like Brucella .

• Conservation across species: The protein is highly conserved among different Brucella species, suggesting that a vaccine targeting YajC might provide cross-protection against multiple Brucella species .

• Diagnostic potential: Current live-attenuated Brucella vaccines interfere with serological diagnostics, making it difficult to distinguish between vaccinated and infected animals . Recombinant subunit vaccines using proteins like YajC could potentially overcome this limitation.

• Safety profile: Recombinant DNA vaccines eliminate many safety concerns associated with live-attenuated vaccines, which cannot be given to pregnant animals without potentially causing abortion .

What methods have been used to clone and express recombinant YajC protein?

The recombinant YajC protein has been cloned and expressed using several methodological approaches:

• Library screening: Researchers identified the yajC gene by screening a gene library of B. abortus 2308 for expression of antigens reacting with immunoglobulin G2a (IgG2a) antibodies from mice vaccinated with B. abortus RB51 .

• Expression system: Both YajC and SecD proteins were expressed in Escherichia coli as fusion proteins with maltose binding protein (MBP) . This expression system utilizes the pMAL vector, which allows for high-level expression and simple purification of fusion proteins.

• Purification: The MBP tag facilitates purification of the fusion protein using amylose resin affinity chromatography, resulting in sufficient quantities of purified recombinant protein for immunological studies .

• Verification: Western blotting was used to confirm the expression and antigenicity of the recombinant proteins, demonstrating that YajC, but not SecD, reacted with immune sera from mice vaccinated with different Brucella strains .

How does the immunogenic profile of YajC compare with other Brucella membrane proteins in experimental models?

The immunogenic profile of YajC differs from other Brucella membrane proteins in several important ways:

What are the molecular mechanisms underlying YajC-induced protective immunity against Brucella infection?

The molecular mechanisms underlying YajC-induced protective immunity against Brucella infection involve several interconnected immunological pathways:

• T cell activation: Recombinant YajC protein stimulates the proliferation of splenocytes from Brucella-vaccinated mice, indicating that YajC-specific T cells are generated during vaccination . These activated T cells likely play a crucial role in orchestrating the immune response against Brucella.

• Th1 polarization: YajC stimulation results in the production of IFN-γ without detectable IL-4, suggesting a polarization toward a Th1-type immune response . This polarization is critical because Th1 responses are essential for controlling intracellular pathogens like Brucella.

• IFN-γ-mediated effector functions: The produced IFN-γ can enhance the microbicidal activities of macrophages, the primary host cells for Brucella, through:

  • Increased production of reactive oxygen and nitrogen species

  • Enhanced phagosome-lysosome fusion

  • Upregulation of MHC class I and II expression, improving antigen presentation

• Antibody production: YajC induces the production of specific antibodies, particularly of the IgG2a subisotype in mice, which is indicative of a Th1-biased response . These antibodies may contribute to protection through mechanisms such as opsonization, enhancing phagocytosis of Brucella by professional phagocytes.

• Memory response: The induction of both cellular and humoral immune responses suggests the establishment of immunological memory, which would be crucial for long-term protection against Brucella infection.

What are the most effective experimental protocols for evaluating YajC-induced immunity in different animal models?

Effective experimental protocols for evaluating YajC-induced immunity in different animal models include:

• Immunization strategies:

  • Recombinant protein vaccination: Purified recombinant YajC protein (either alone or as a fusion protein) administered with appropriate adjuvants

  • DNA vaccination: Plasmid DNA encoding YajC, which can induce both humoral and cell-mediated immune responses

  • Prime-boost strategies: Combining different vaccination approaches (e.g., DNA prime, protein boost) to enhance immune responses

• Assessment of humoral immunity:

  • ELISA for detection of YajC-specific antibodies in serum

  • Western blotting to confirm antibody specificity and cross-reactivity

  • Isotype analysis to determine the IgG subclass distribution (IgG1 vs. IgG2a in mice), which provides insights into the Th1/Th2 balance

• Evaluation of cell-mediated immunity:

  • Lymphocyte proliferation assays using splenocytes or peripheral blood mononuclear cells stimulated with recombinant YajC

  • Cytokine profiling, particularly measuring IFN-γ, IL-4, and other relevant cytokines using ELISA or intracellular cytokine staining

  • Flow cytometry analysis of T cell subsets (CD4+ vs. CD8+) and their activation status

• Challenge studies:

  • Infection with virulent Brucella strains (appropriate to the animal model) after vaccination

  • Assessment of bacterial load in target organs (spleen, liver, lymph nodes) at different time points post-challenge

  • Histopathological examination of affected tissues

• Species-specific considerations:

  • Mice: Commonly used for initial evaluation, with BALB/c mice being particularly susceptible to Brucella infection

  • Goats: Natural hosts for B. melitensis, allowing assessment of protection against abortion and other clinical manifestations

  • Cattle: Natural hosts for B. abortus, important for evaluating cross-protection

How do genetic variations in the yajC gene among different Brucella species impact its immunogenic potential?

The impact of genetic variations in the yajC gene among different Brucella species on its immunogenic potential is a critical consideration for vaccine development:

• Sequence conservation: The main Brucella species (B. abortus, B. melitensis, and B. suis) share approximately 99% identity at the DNA level, suggesting high conservation of the yajC gene across species . This high degree of conservation likely contributes to the observed cross-reactivity of immune responses against YajC from different Brucella species .

• Epitope preservation: The conservation of amino acid sequences corresponding to immunodominant epitopes is particularly important for maintaining cross-protective immunity. The selection of conserved epitopes ranging from 25 to 70 amino acids in length has been used in developing recombinant DNA vaccines against multiple Brucella species .

• Expression regulation: While the coding sequence may be highly conserved, variations in promoter regions or regulatory elements might affect the expression levels of YajC in different Brucella species under various conditions. Evidence suggests that YajC is expressed at low levels under normal growth conditions but may be upregulated during infection .

• Post-translational modifications: Potential differences in post-translational modifications of YajC among Brucella species could affect protein folding, stability, and presentation to the immune system, potentially impacting immunogenicity.

• Cellular localization: Minor variations in the signal sequence or transmembrane domains could alter the cellular localization or membrane insertion of YajC, affecting its accessibility to the immune system during infection.

What are the challenges and solutions in designing multi-epitope vaccines incorporating YajC peptides?

Designing multi-epitope vaccines incorporating YajC peptides presents several challenges and potential solutions:

Challenges:

• Epitope identification: Identifying the most immunogenic and protective epitopes within the YajC protein requires extensive computational analysis and experimental validation .

• Epitope optimization: Determining the optimal length (25-70 amino acids) and sequence of epitopes to maximize immunogenicity while maintaining proper folding and presentation .

• Delivery systems: Selecting appropriate delivery platforms (DNA vaccines, recombinant protein subunits, viral vectors) that can effectively present multiple epitopes to the immune system.

• Adjuvant selection: Identifying adjuvants that can enhance the Th1-biased immune response required for protection against Brucella without causing excessive reactogenicity.

• Interference between epitopes: Multiple epitopes may compete for immune recognition or interfere with each other's processing and presentation.

Solutions:

• In silico approaches: Using bioinformatics tools to predict B and T cell epitopes, epitope accessibility, and potential cross-reactivity with host proteins .

• Rational epitope design: Incorporating both B and T cell epitopes, ensuring appropriate spacing between epitopes, and including promiscuous epitopes that can bind to multiple MHC molecules to increase population coverage.

• Modular vaccine design: Constructing vaccines with interchangeable modules containing epitopes from different Brucella proteins, including YajC, to induce broader immune responses.

• Novel adjuvant formulations: Testing combinations of adjuvants, such as CpG oligonucleotides, that can specifically promote Th1-type immune responses .

• Prime-boost strategies: Employing heterologous prime-boost immunization regimens using different delivery platforms to maximize epitope-specific immune responses.

• Diagnostic compatibility: Designing vaccines that exclude epitopes used in diagnostic tests, allowing for differentiation between vaccinated and infected animals (DIVA strategy) .

What are the optimal methods for purifying recombinant YajC protein while maintaining its immunogenic properties?

The optimal methods for purifying recombinant YajC protein while preserving its immunogenic properties involve several strategic approaches:

• Expression systems:

  • E. coli-based expression using the pMAL vector system to produce YajC as a fusion protein with maltose binding protein (MBP)

  • Alternative expression hosts like Pichia pastoris may be considered for proteins requiring eukaryotic post-translational modifications

• Purification strategies:

  • Affinity chromatography using amylose resin for MBP-tagged YajC fusion proteins

  • Immobilized metal affinity chromatography (IMAC) for His-tagged variants

  • Size exclusion chromatography as a polishing step to achieve higher purity

• Protein solubility considerations:

  • As a membrane protein, YajC may have hydrophobic regions that reduce solubility

  • Use of solubility-enhancing fusion partners (MBP, SUMO, thioredoxin) can improve expression and solubility

  • Careful selection of buffer conditions (pH, ionic strength, presence of mild detergents) to maintain protein in solution

• Preservation of structural integrity:

  • Avoiding harsh elution conditions that might denature the protein

  • Including stabilizing agents such as glycerol or specific detergents in storage buffers

  • Minimizing freeze-thaw cycles by appropriate aliquoting

• Quality control methods:

  • SDS-PAGE and Western blotting to confirm purity and immunoreactivity

  • Circular dichroism spectroscopy to assess secondary structure integrity

  • Functional assays such as immunoreactivity with sera from Brucella-infected animals to confirm preservation of epitopes

How can researchers effectively evaluate the cellular immune response to YajC in laboratory and target animal species?

Researchers can effectively evaluate the cellular immune response to YajC using a combination of in vitro and in vivo approaches:

• Lymphocyte proliferation assays:

  • Isolation of peripheral blood mononuclear cells (PBMCs) or splenocytes from immunized animals

  • Stimulation with purified recombinant YajC protein or YajC-derived peptides

  • Measurement of proliferation using [³H]-thymidine incorporation or dye-based methods such as CFSE dilution

  • Analysis at multiple time points (e.g., 3-day and 5-day cultures) to capture different kinetics of response

• Cytokine profiling:

  • ELISA-based detection of secreted cytokines (IFN-γ, IL-4, IL-2, TNF-α) in culture supernatants

  • Intracellular cytokine staining combined with flow cytometry to identify cytokine-producing cell subsets

  • ELISpot assays to enumerate cytokine-producing cells at the single-cell level

  • Quantitative RT-PCR for cytokine mRNA expression analysis

• T cell phenotyping:

  • Flow cytometric analysis of T cell activation markers (CD25, CD69, HLA-DR)

  • Characterization of memory T cell subsets (effector, central, tissue-resident memory)

  • Assessment of T cell exhaustion markers (PD-1, CTLA-4, LAG-3) following repeated stimulation

• Antigen-specific T cell detection:

  • MHC tetramer or pentamer staining to enumerate antigen-specific T cells

  • Adoptive transfer experiments using labeled T cells to track in vivo responses

  • T cell receptor (TCR) sequencing to characterize the diversity of the responding T cell repertoire

• In vivo challenge models:

  • Delayed-type hypersensitivity (DTH) reactions following intradermal injection of YajC

  • Assessment of protection against virulent Brucella challenge in vaccinated animals

  • Correlation of cellular immune parameters with protection levels

What bioinformatic approaches can identify potential T and B cell epitopes within the YajC protein sequence?

Several bioinformatic approaches can be employed to identify potential T and B cell epitopes within the YajC protein sequence:

• T cell epitope prediction:

  • MHC binding prediction algorithms (NetMHC, IEDB Analysis Resource, SYFPEITHI) to identify peptides likely to bind MHC class I and II molecules

  • Proteasomal cleavage site prediction to identify peptides likely to be generated during antigen processing

  • TAP binding prediction to assess transport efficiency of peptides into the endoplasmic reticulum

  • Integrated approaches combining multiple prediction methods to increase accuracy

• B cell epitope prediction:

  • Linear (continuous) epitope prediction based on properties such as hydrophilicity, flexibility, accessibility, and antigenic propensity

  • Conformational (discontinuous) epitope prediction using 3D structural information or homology models

  • Sequence-based tools like BepiPred, ABCpred, and Ellipro

  • Evolutionary analysis to identify conserved surface-exposed regions

• Structural biology approaches:

  • Homology modeling of YajC structure based on related proteins with known structures

  • Molecular dynamics simulations to assess epitope accessibility and flexibility

  • Surface mapping of predicted epitopes on 3D models to visualize potential antibody binding sites

• Comparative genomics:

  • Sequence alignment across Brucella species to identify conserved regions suitable for cross-protective immunity

  • Epitope conservation analysis to prioritize epitopes present in multiple strains and species

  • Exclusion of epitopes with significant homology to host proteins to reduce potential autoimmunity risks

• Experimental validation strategies:

  • Epitope mapping using overlapping peptide libraries

  • ELISA or peptide microarray screening using sera from infected or vaccinated animals

  • In vitro T cell stimulation assays with synthetic peptides corresponding to predicted epitopes

How can researchers develop standardized immunoassays for detecting YajC-specific antibodies in vaccinated or infected animals?

Developing standardized immunoassays for detecting YajC-specific antibodies requires systematic approaches to ensure reliability and reproducibility:

• Antigen preparation:

  • Production of highly pure recombinant YajC protein using consistent expression and purification protocols

  • Characterization of protein quality using SDS-PAGE, Western blot, and mass spectrometry

  • Determination of optimal antigen concentration through titration experiments

  • Consideration of different forms of the antigen (full-length protein, specific domains, or synthetic peptides)

• ELISA development:

  • Optimization of coating conditions (buffer, pH, temperature, time)

  • Selection of appropriate blocking agents to minimize background

  • Determination of optimal sample dilutions and incubation conditions

  • Selection of detection antibodies with high specificity for target species immunoglobulins

  • Establishment of standard curves using reference sera with known antibody titers

• Western blot protocols:

  • Standardization of protein transfer conditions to ensure consistent antigen presentation

  • Optimization of membrane blocking to reduce non-specific binding

  • Selection of detection systems (colorimetric, chemiluminescent, fluorescent) based on required sensitivity

  • Development of densitometric methods for semi-quantitative analysis

• Validation parameters:

  • Analytical sensitivity (limit of detection)

  • Analytical specificity (cross-reactivity with antibodies against related proteins)

  • Diagnostic sensitivity and specificity using panels of sera from infected, vaccinated, and naive animals

  • Repeatability (intra-assay variation) and reproducibility (inter-assay and inter-laboratory variation)

  • Stability of reagents and test performance over time

• Reference standards:

  • Development of species-specific positive and negative control sera

  • Establishment of international reference standards through collaborative studies

  • Creation of calibration curves for quantitative antibody measurement

What are the critical parameters for designing challenge studies to evaluate YajC-based vaccine efficacy against Brucella infection?

Critical parameters for designing challenge studies to evaluate YajC-based vaccine efficacy against Brucella infection include:

• Animal model selection:

  • Natural host species (cattle, goats, sheep) for translational relevance

  • Laboratory animal models (mice) for preliminary screening and mechanism studies

  • Consideration of age, sex, genetic background, and immune status of the animals

  • Sample size calculation based on expected effect size and statistical power

• Vaccination protocol:

  • Dose optimization through dose-response studies

  • Route of administration (subcutaneous, intramuscular, intradermal, mucosal)

  • Prime-boost strategy (interval between doses, homologous vs. heterologous boosting)

  • Adjuvant selection based on the desired type of immune response (Th1-biased for Brucella)

  • Timing of immune response assessment pre-challenge

• Challenge protocol:

  • Selection of the challenge strain (virulence, species relevance, dose)

  • Route of challenge (natural route of infection when possible)

  • Timing of challenge relative to vaccination (to assess short and long-term protection)

  • Biosafety considerations and containment requirements

• Outcome measures:

  • Bacterial load quantification in target organs (spleen, liver, lymph nodes)

  • Clinical scoring systems for disease manifestations

  • Reproductive outcomes in natural hosts (abortion rates, fertility)

  • Serological responses (differentiating infection from vaccination if possible)

  • Correlation of immune parameters with protection

• Control groups:

  • Unvaccinated challenged controls to confirm successful infection

  • Gold standard vaccine controls (e.g., B. abortus RB51, B. melitensis Rev.1) for comparative efficacy

  • Sham-vaccinated controls to assess adjuvant effects

  • Unchallenged controls to establish baseline parameters

What novel delivery systems could enhance the immunogenicity and efficacy of YajC-based vaccines?

Several novel delivery systems hold promise for enhancing the immunogenicity and efficacy of YajC-based vaccines:

• Nanoparticle-based delivery:

  • Biodegradable polymer nanoparticles (PLGA, PCL) that can provide sustained release of YajC protein

  • Liposomal formulations that can enhance antigen uptake by antigen-presenting cells

  • Virus-like particles (VLPs) displaying YajC epitopes on their surface

  • Self-assembling protein nanoparticles that can present YajC in a highly immunogenic multimeric form

• Nucleic acid-based platforms:

  • DNA vaccines encoding YajC, optimized for expression in mammalian cells

  • mRNA vaccines with modified nucleosides for enhanced stability and translation efficiency

  • Self-amplifying RNA vaccines for prolonged antigen expression

  • Prime-boost strategies combining nucleic acid and protein-based vaccines

• Viral vector systems:

  • Replication-deficient adenoviral vectors expressing YajC

  • Modified vaccinia Ankara (MVA) or other poxvirus vectors

  • Vesicular stomatitis virus (VSV) or other RNA virus-based vectors

  • Bacterial vectors such as attenuated Salmonella expressing YajC

• Mucosal delivery strategies:

  • Oral delivery systems protected from gastric degradation

  • Intranasal formulations with appropriate mucosal adjuvants

  • Pulmonary delivery for inducing respiratory tract immunity

  • Microencapsulation techniques to protect antigens during mucosal transit

• Novel adjuvant combinations:

  • TLR agonists particularly targeting TLR9 (CpG oligonucleotides) for enhanced Th1 responses

  • Saponin-based adjuvants like ISCOMS or QS-21

  • Combination adjuvant systems like AS01 or AS02

  • Cytokine adjuvants (IL-12, GM-CSF) to direct specific immune responses

How might YajC be incorporated into multi-component vaccines targeting multiple Brucella virulence factors?

YajC could be strategically incorporated into multi-component vaccines targeting multiple Brucella virulence factors through several approaches:

What are the prospects for developing DIVA (Differentiating Infected from Vaccinated Animals) vaccines based on YajC technology?

The prospects for developing DIVA vaccines based on YajC technology are promising and address a critical need in brucellosis control:

• Current diagnostic limitations:

  • Existing live-attenuated Brucella vaccines (B. abortus RB51, B. melitensis Rev.1) interfere with serological diagnostics, making it difficult to distinguish between vaccinated and infected animals

  • This limitation hampers surveillance and control programs in endemic regions

• YajC-based DIVA vaccine strategies:

  • Subunit vaccines using recombinant YajC would not contain the full complement of Brucella antigens used in diagnostic tests

  • Modified YajC constructs with deletion or modification of conserved epitopes used in diagnostic assays

  • Addition of unique epitope tags to vaccine-derived YajC to allow detection of vaccine-specific antibodies

• Complementary diagnostic approaches:

  • Development of paired diagnostic tests detecting different antigens than those included in the vaccine

  • Epitope-specific serological assays that can distinguish natural infection from vaccination

  • PCR-based methods to detect field strain-specific genetic sequences absent in vaccine strains

  • Differential cytokine signature analysis (e.g., IFN-γ release assays with specific antigens)

• Validation requirements:

  • Extensive testing in target species under field conditions

  • Evaluation of diagnostic performance at different time points post-vaccination

  • Assessment of cross-reactivity with other pathogens that may cause serological interference

  • Determination of appropriate diagnostic cut-off values for differentiation

• Implementation considerations:

  • Regulatory approval pathways for both the vaccine and companion diagnostic tests

  • Cost-effectiveness analysis compared to current control strategies

  • Training requirements for field veterinarians and laboratory personnel

  • Integration with existing surveillance systems and eradication programs

How can systems biology and computational modeling accelerate YajC-based vaccine development?

Systems biology and computational modeling can significantly accelerate YajC-based vaccine development through several innovative approaches:

• Immune response prediction:

  • In silico modeling of antigen processing and presentation pathways

  • Prediction of how YajC epitopes interact with host immune receptors

  • Simulation of immune response kinetics following vaccination

  • Network analysis of gene expression patterns to predict protective vs. non-protective responses

• Epitope optimization:

  • Computational design of YajC constructs with enhanced MHC binding and immunogenicity

  • Structure-based optimization of B cell epitopes for improved antibody recognition

  • Prediction of epitope dominance hierarchies to guide vaccine design

  • Population coverage analysis across different MHC haplotypes

• Adjuvant selection and formulation:

  • Modeling of adjuvant-antigen interactions and their effects on immunogenicity

  • Prediction of optimal adjuvant combinations for specific immune response profiles

  • Simulation of antigen release kinetics from different delivery systems

  • Virtual screening of novel adjuvant compounds

• Integration of multi-omics data:

  • Transcriptomic analysis to understand host responses to YajC vaccination

  • Proteomic profiling to identify correlates of protection

  • Metabolomic studies to characterize metabolic signatures associated with protective immunity

  • Integration of multiple data types through machine learning approaches

• Translational modeling:

  • Allometric scaling to translate dosing from animal models to humans

  • Physiologically-based pharmacokinetic (PBPK) modeling of vaccine distribution

  • Prediction of immune response variability across different populations

  • Simulation of vaccination strategies at the population level

What are the potential applications of YajC-based immunity beyond prophylactic vaccination against brucellosis?

YajC-based immunity has potential applications beyond prophylactic vaccination against brucellosis:

• Therapeutic vaccination:

  • Treatment of chronic or persistent Brucella infections through immune stimulation

  • Combination with antibiotics to enhance clearance of the pathogen

  • Reduction of disease severity and complications in already infected individuals

  • Prevention of relapse following conventional treatment

• Diagnostic platforms:

  • Development of YajC-based serological assays for improved brucellosis diagnosis

  • Creation of point-of-care diagnostic tests suitable for field use in resource-limited settings

  • Multiplex diagnostic arrays incorporating YajC along with other Brucella antigens

  • Biomarker discovery based on YajC-specific immune responses

• Immunomodulatory applications:

  • Investigation of YajC as a potential carrier protein for conjugate vaccines against other pathogens

  • Exploitation of YajC's Th1-biasing properties for therapeutic applications in diseases requiring Th1 immunity

  • Study of YajC-induced signaling pathways for understanding fundamental immunological mechanisms

  • Development of YajC-derived peptides as immunomodulatory agents

• Vector vaccine platforms:

  • Use of YajC epitopes in conjunction with established vector platforms (viral, bacterial)

  • Development of multivalent vaccines expressing YajC along with antigens from other pathogens common in livestock

  • Creation of chimeric antigens incorporating protective elements from YajC and other pathogen proteins

• Research tools:

  • YajC-specific antibodies as tools for studying Brucella pathogenesis

  • YajC expression systems as models for membrane protein production and purification

  • Structure-function studies of YajC to understand protein secretion mechanisms in bacteria

  • Investigation of YajC homologs in other bacterial pathogens as potential vaccine candidates