Recombinant Salmonella gallinarum Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is Recombinant Salmonella gallinarum Probable ubiquinone biosynthesis protein UbiB?

Recombinant Salmonella gallinarum UbiB is a full-length protein (546 amino acids) produced through heterologous expression systems, typically E. coli, for research purposes. The native protein functions as a probable protein kinase involved in ubiquinone (coenzyme Q) biosynthesis within Salmonella gallinarum strain 287/91 (NCTC 13346). The recombinant version is commonly produced with an N-terminal histidine tag to facilitate purification and detection in laboratory settings. This protein is classified as a transmembrane protein and plays a role in the electron transport chain and cellular respiration processes . The recombinant form allows researchers to study the protein's biochemical properties, structure, and function outside the native bacterial context.

How is recombinant UbiB typically produced for research purposes?

Production of recombinant Salmonella gallinarum UbiB follows standard molecular biology protocols with specific optimizations:

• Expression System Selection: Most commonly produced in E. coli expression systems, as indicated in the product specifications . This heterologous system allows for high-yield protein expression under controllable conditions.

• Vector Construction: The full-length ubiB gene (nucleotides encoding amino acids 1-546) is cloned into an expression vector containing:

  • An N-terminal histidine tag sequence (typically 10xHis)

  • Appropriate promoter (often T7 or similar inducible promoter)

  • Selection markers for transformation verification

• Transformation and Expression: The recombinant vector is transformed into E. coli expression strains (commonly BL21(DE3) or derivatives). Expression is induced using IPTG or other inducers depending on the vector design.

• Purification Process:

  • Bacterial lysis (sonication or chemical methods)

  • Affinity chromatography using Ni-NTA or similar matrices that bind the His-tag

  • Elution with imidazole-containing buffers

  • Buffer exchange to remove imidazole

• Product Formulation: The purified protein is typically provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, available in either liquid form or as a lyophilized powder .

What are the storage and stability considerations for recombinant UbiB?

Proper storage of recombinant Salmonella gallinarum UbiB is crucial for maintaining its structural integrity and biological activity:

For working with the protein:

• Briefly centrifuge vials before opening to bring contents to the bottom

• Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• For working aliquots, store at 4°C for up to one week

The shelf life depends on multiple factors including buffer composition, storage temperature, and the intrinsic stability of the protein itself. Trehalose (6%) is included in the formulation buffer to enhance stability during freeze-thaw cycles.

What molecular techniques can be used to study UbiB function in Salmonella gallinarum?

Several advanced molecular approaches can be employed to elucidate UbiB function:

• Gene Knockout Studies:

  • λ-Red recombination technology allows for precise deletion of the ubiB gene

  • Creation of isogenic markerless knockout mutants permits direct comparison with wild-type strains

  • PCR verification using specific primers flanking the ubiB region confirms successful knockouts

• Complementation Assays:

  • Reintroduction of functional ubiB on plasmids to knockout strains

  • Analysis of phenotype restoration confirms gene function

  • Use of inducible promoters allows titration of expression levels

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation using antibodies against the His-tag

  • Bacterial two-hybrid systems to identify interaction partners

  • Cross-linking studies followed by mass spectrometry

• Enzymatic Activity Assays:

  • In vitro kinase assays using purified recombinant UbiB

  • Analysis of ubiquinone biosynthesis pathway intermediates by HPLC

  • Competitive inhibition studies to map the active site

• Structural Biology Approaches:

  • X-ray crystallography of purified UbiB

  • Cryo-EM for membrane-associated conformations

  • Computational modeling based on homologous proteins

How does genetic manipulation of Salmonella gallinarum, such as UbiB modification, affect bacterial pathogenicity?

While the search results don't directly address UbiB deletion effects, they provide insights into genetic engineering approaches in Salmonella Gallinarum that can be applied to UbiB research:

• Attenuation Strategies:
  The purB gene deletion in Salmonella Gallinarum demonstrates how targeted gene disruption can attenuate virulence. This approach using λ-Red recombination technology could be applied to ubiB .

• Metabolic Impact Assessment:

  • Growth rate comparisons between wild-type and knockout strains

  • UbiB deletion would likely affect ubiquinone synthesis, impacting the electron transport chain

  • Supplementation experiments could determine if external metabolites restore growth

• Virulence Evaluation Protocol:
  Based on the purB study methodology, UbiB function could be assessed through:

  • In vivo pathogenicity testing in 3-day-old chickens

  • Monitoring mortality rates, clinical signs, and lesion scores

  • Bacterial persistence in tissues (e.g., liver, spleen) over time

  • Clearance rate determination via tissue sampling and PCR detection

• Quantitative Assessment:
  The following parameters would provide measurable outcomes:

  • Reduction in growth rate compared to wild-type (purB deletion showed 66.5% reduction)

  • Clinical sign scores (on a defined scale, e.g., 0-3)

  • Lesion scores in affected tissues

  • Bacterial load quantified as log10 CFU/g of tissue

What are the potential applications of recombinant UbiB and UbiB mutants in vaccine development?

Recombinant UbiB protein and engineered Salmonella strains with ubiB modifications have several potential vaccine applications:

• Live Attenuated Vaccine Development:

  • Creation of Salmonella gallinarum strains with ubiB deletion or modification

  • Safety assessment through pathogenicity studies in target species

  • Evaluation of immune response and protection against challenge

• Subunit Vaccine Approaches:

  • Use of purified recombinant UbiB as a vaccine antigen

  • Formulation with appropriate adjuvants

  • Delivery system optimization for poultry vaccination

• Comparative Advantage Assessment:
  Based on the purB knockout study, a ubiB-modified live attenuated vaccine might offer:

  • Reduced clinical signs compared to wild-type infection

  • Efficient clearance from tissues (14 days post-inoculation)

  • Zero mortality compared to wild-type challenge

• Cross-Protection Potential:

  • Evaluation against different Salmonella serovars

  • Assessment of immune response using serological tests like Rapid Plate Agglutination

• Regulatory Considerations:

  • All recombinant DNA research, including vaccine development with modified Salmonella strains, must comply with NIH Guidelines

  • IBC (Institutional Biosafety Committee) approval required prior to starting projects

  • Additional requirements for non-exempt work

How can researchers verify the functional integrity of recombinant UbiB?

Verifying functional integrity of recombinant Salmonella gallinarum UbiB requires multi-faceted approaches:

• Structural Integrity Assessment:

  • SDS-PAGE analysis to confirm protein size (>90% purity standard)

  • Western blotting using anti-His antibodies or specific anti-UbiB antibodies

  • Circular dichroism spectroscopy to evaluate secondary structure elements

  • Limited proteolysis to assess proper folding

• Enzymatic Activity Verification:

  • Kinase activity assays using potential substrates

  • ATP binding and hydrolysis measurements

  • Monitoring ubiquinone production in complementation assays

• Functional Complementation:

  • Introduction of recombinant UbiB into UbiB-deficient strains

  • Restoration of growth in minimal media

  • Recovery of ubiquinone biosynthesis pathway function

• Binding Partner Interactions:

  • Surface plasmon resonance to quantify interactions with known partners

  • Pull-down assays using the His-tag to identify in vivo binding partners

  • Fluorescence polarization for ligand binding studies

How does UbiB function compare across different Salmonella species and other bacteria?

Comparative analysis of UbiB across bacterial species provides evolutionary and functional insights:

Research approaches for comparative studies:

• Sequence alignment and phylogenetic analysis of UbiB proteins

• Expression of heterologous UbiB proteins in a common host

• Cross-complementation studies between species

• Metabolic profiling of ubiquinone biosynthesis across species