Recombinant Brucella melitensis biotype 2 Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the role of Succinyl-CoA ligase in Brucella melitensis metabolism?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) is likely part of the TCA cycle in B. melitensis, similar to the dihydrolipoamide succinyltransferase (SucB) that was identified as an immunogenic protein in infected sheep. The SucB protein is an enzyme of the α-ketoglutarate dehydrogenase complex involved in energy metabolism . SucC would function in a related pathway, catalyzing the conversion of succinyl-CoA to succinate while generating ATP, maintaining essential metabolic functions for bacterial survival.

How does sequence conservation of Brucella recombinant proteins affect their research applications?

Based on research with related proteins, we can infer that sucC likely shows significant sequence conservation among Brucella species. For example, the amino acid sequence of SucB from B. melitensis showed 88.8% identity to the homologous protein from B. abortus and 51.2% identity to that from E. coli . This conservation pattern is important because:

• High conservation within Brucella species suggests potential cross-protection when used as vaccine candidates

• Moderate conservation with other bacterial species requires careful specificity testing for diagnostic applications

• Sequence variation may identify Brucella-specific epitopes useful for developing targeted diagnostics

What are the immunological characteristics of Brucella recombinant proteins in host responses?

Recombinant Brucella proteins often elicit significant immune responses. For example, recombinant Omp31 induced a vigorous immunoglobulin G (IgG) response with higher IgG1 than IgG2 titers. Additionally, spleen cells from rOmp31-immunized mice produced interleukin 2 (IL-2) and gamma interferon, but not IL-10 or IL-4 after in vitro stimulation, suggesting the induction of a T helper 1 (Th1) response . Splenocytes from rOmp31-vaccinated animals also demonstrated specific cytotoxic-T-lymphocyte activity . Similar patterns may be expected for other immunogenic Brucella proteins like sucC.

What expression systems are most effective for producing recombinant B. melitensis proteins?

Based on published research with Brucella recombinant proteins, the following systems have proven effective:

For sucC specifically, starting with the E. coli BL21(DE3) system with a pET vector would be recommended based on success with other Brucella proteins.

What purification strategies yield the highest purity and activity for Brucella recombinant proteins?

Recombinant Brucella proteins often require specific purification approaches:

• For insoluble proteins in inclusion bodies (common with Brucella proteins):

  • Extraction under denaturing conditions using 8M urea

  • Ni-NTA affinity chromatography for His-tagged proteins

  • Refolding protocols post-purification to restore activity

• For yield assessment:

  • Protein quantification methods like Bradford assay (VirB12 yield was approximately 0.6 mg/ml of culture)

  • SDS-PAGE and Western blot confirmation of purity and immunoreactivity

How can researchers optimize codon usage for improved expression of B. melitensis genes in heterologous systems?

While not explicitly discussed in the search results, codon optimization is important for heterologous expression of Brucella proteins:

• Analyze codon usage bias in B. melitensis compared to expression host (typically E. coli)

• Optimize rare codons to match preferred codons of expression host

• Adjust GC content while maintaining codon optimization

• Consider using specialized E. coli strains supplying rare tRNAs if codon optimization isn't feasible

What animal models and protocols are most appropriate for evaluating B. melitensis recombinant proteins?

Based on established protocols for B. melitensis vaccine candidates:

• Mouse model specifications:

  • 6-8 week-old female BALB/c mice have been successfully used

  • Random distribution into experimental groups with proper housing conditions

  • Water and food provided ad libitum

• Immunization protocol:

  • Protein administration with appropriate adjuvant (e.g., incomplete Freund's adjuvant)

  • Typical schedule: injections on days 0 and 15

  • Control groups: PBS in adjuvant (negative) and killed B. melitensis (positive)

• Sample collection:

  • Serum collection at multiple timepoints (e.g., days 15, 30, 45, 60, and 75 post-immunization)

  • Sacrifice for immune response analysis or challenge with virulent strains

What methods effectively evaluate protective immunity induced by Brucella recombinant proteins?

Protection studies should include these essential components:

• Challenge protocol:

  • Intravenous injection with virulent Brucella strains (e.g., 10^4 CFU of B. melitensis H38S)

  • Sacrifice 30 days post-challenge

• Protection assessment:

  • Bacterial load determination in spleen and liver

  • Serial dilution and plating of tissue homogenates

  • CFU counting and calculation of mean log CFU ± standard deviation per group

  • Log units of protection calculation by subtracting mean log CFU of vaccinated group from control group

• Immunological correlates:

  • Cytokine production profiling (IFN-γ, IL-2, IL-4, IL-10)

  • Antibody isotype analysis (IgG1 vs. IgG2)

  • T cell response characterization (CD4+ vs. CD8+)

How should epitope mapping be performed for identifying immunodominant regions of B. melitensis proteins?

Based on successful approaches with Omp31:

• Peptide design approach:

  • Generate overlapping peptides spanning the entire protein sequence

  • Test synthetic peptides of various lengths (e.g., 27-aa peptide from Omp31)

  • Focus on predicted surface-exposed regions

• Immunological testing:

  • In vitro stimulation of splenocytes from immunized animals with candidate peptides

  • Cytokine production measurement

  • Antibody binding assays

• Protection assessment:

  • Immunize with individual peptides using the same protocol as whole protein

  • Challenge with virulent strains

  • Compare protection levels to whole protein immunization (noted that a 27-aa peptide from Omp31 induced protection similar to whole recombinant protein against B. melitensis)

What performance parameters should be evaluated when developing diagnostic tests based on recombinant B. melitensis proteins?

Based on the VirB12 study, a comprehensive evaluation includes:

These parameters should be determined by comparing results against a reference standard (e.g., commercial ELISA) and using well-characterized serum panels.

How can researchers address cross-reactivity issues when developing serological tests based on Brucella recombinant proteins?

Cross-reactivity is a critical consideration, especially given the LPS cross-reactivity issues in current brucellosis diagnostics:

• Specificity testing against related bacteria:

  • Test against closely related alphaproteobacteria (Ochrobactrum, Phyllobacterium, Rhizobium, Agrobacterium)

  • Include common bacterial pathogens causing similar clinical presentations

• Serum panel composition:

  • Include sera from patients with confirmed infections by other pathogens

  • Test with sera from healthy individuals from endemic and non-endemic regions

• Comparative analysis:

  • Direct comparison with traditional LPS-based tests to demonstrate improved specificity

  • ROC curve analysis to determine optimal cutoff values

What ELISA formats are most effective for Brucella recombinant protein-based diagnostics?

For developing an ELISA based on recombinant Brucella proteins like sucC:

• Indirect ELISA protocol:

  • Coat plates with purified recombinant protein at optimized concentration

  • Block with appropriate blocking agent (e.g., BSA)

  • Test with serum dilutions to determine optimal working concentration

  • Detect with species-appropriate secondary antibody conjugated to enzyme

  • Visualize with substrate like TMB and measure absorbance

• Protocol optimization:

  • Titrate antigen coating concentration

  • Optimize serum dilution (typically 1:1000 for Western blot)

  • Determine optimal incubation times and temperatures

How can researchers overcome protein solubility issues when expressing Brucella recombinant proteins?

Many Brucella recombinant proteins form inclusion bodies, as observed with VirB12 :

• Solubility enhancement strategies:

  • Lower induction temperature (16-25°C)

  • Reduce IPTG concentration (0.1-0.5 mM instead of 1 mM)

  • Co-express molecular chaperones

  • Use solubility tags (MBP, SUMO, GST)

• Extraction approaches for inclusion bodies:

  • Optimize lysis conditions

  • Use denaturing agents (8M urea successfully used for VirB12)

  • Develop refolding protocols to restore native conformation

What are the critical factors in designing primers for cloning Brucella genes?

Based on successful cloning of Brucella genes:

• Primer design considerations:

  • Include appropriate restriction sites compatible with expression vector

  • Add extra bases (3-6) outside restriction sites for efficient enzyme cutting

  • Check for internal restriction sites within the gene

  • Consider adding His-tag sequence if not provided by vector

• PCR optimization:

  • Use high-fidelity DNA polymerase to minimize errors

  • Optimize annealing temperature based on primer Tm values

  • Consider GC content of Brucella genome (generally high)

How can protein structure-function relationships be determined for novel Brucella recombinant proteins?

For characterizing novel proteins like sucC:

• Computational approaches:

  • Homology modeling based on related proteins with known structures

  • Functional domain prediction

  • Active site identification

• Experimental validation:

  • Site-directed mutagenesis of predicted functional residues

  • Enzymatic activity assays (particularly relevant for metabolic enzymes like sucC)

  • Protein-protein interaction studies