Recombinant Salmonella typhimurium Bifunctional protein aas (aas)

What is Bifunctional protein aas in Salmonella typhimurium?

Bifunctional protein aas in Salmonella typhimurium is a 719-amino acid protein (UniProt ID: Q8ZMA4) that likely plays multiple functional roles in bacterial metabolism and potentially in pathogenesis. The "bifunctional" designation indicates it performs dual biochemical functions, which is common for proteins involved in bacterial metabolic pathways. Based on sequence analysis, it appears to contain domains associated with acyl-CoA synthetase activity and potentially phospholipid metabolism. When expressed recombinantly, it can be produced with an N-terminal His tag in E. coli expression systems .

What is the molecular structure and characteristics of Bifunctional protein aas?

The Bifunctional protein aas consists of 719 amino acids with several functional domains. Analysis of its amino acid sequence reveals potential substrate binding sites and catalytic domains. The protein contains motifs suggesting enzymatic activity, particularly in lipid metabolism pathways. The complete amino acid sequence (MLFGFFRNLFRVLYRVRVTGDVRALQGNRVLITPNHVSFIDGMLLALFLPVRPVFAVYTS...) indicates the presence of hydrophobic regions and potential membrane association domains . The protein can be produced in E. coli expression systems and purified to greater than 90% purity using appropriate chromatographic techniques, yielding a stable protein that can be stored as a lyophilized powder .

How does Bifunctional protein aas compare between Salmonella typhimurium and Salmonella choleraesuis?

Both Salmonella typhimurium and Salmonella choleraesuis express Bifunctional protein aas with similar characteristics. The proteins from both species are 719 amino acids in length and can be expressed recombinantly with N-terminal His tags in E. coli expression systems . While the exact sequence homology is not specified in the search results, proteins from these closely related Salmonella species likely share high sequence similarity and conserved functional domains. Both can be purified using similar methodologies and maintained in similar buffer conditions (Tris/PBS-based buffer with 6% Trehalose at pH 8.0) . Research approaches for studying either protein would follow similar protocols, though specific antibodies might be needed to distinguish between them in experimental settings.

What expression systems yield optimal results for Recombinant S. typhimurium Bifunctional protein aas?

E. coli has been established as an effective expression system for Recombinant S. typhimurium Bifunctional protein aas. The protein has been successfully expressed with an N-terminal His tag in E. coli, indicating this system provides suitable conditions for proper protein folding and expression . For optimal expression, researchers should consider using T7 promoter-based vectors in BL21(DE3) E. coli strains or derivatives. Induction conditions should be optimized, typically using IPTG at concentrations between 0.1-1.0 mM when the culture reaches OD600 of 0.6-0.8. Lower temperatures during induction (16-25°C) often improve protein solubility. Alternative approaches may include using specialized strains like Rosetta to address potential codon bias issues or SHuffle/Origami strains if disulfide bonds are critical for protein folding.

What purification strategies yield the highest purity for Recombinant S. typhimurium Bifunctional protein aas?

Given that Recombinant S. typhimurium Bifunctional protein aas is produced with an N-terminal His tag, immobilized metal affinity chromatography (IMAC) serves as the primary purification method . To achieve >90% purity as reported in the specifications, a multi-step purification protocol is recommended:

• Initial IMAC purification:

  • Ni-NTA or Co-based resins with imidazole gradient elution (20-250 mM)

  • Buffer optimization (pH 7.5-8.0, 150-300 mM NaCl) to maintain stability

• Secondary purification:

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography based on the protein's isoelectric point

Quality control should include SDS-PAGE analysis, Western blotting with anti-His antibodies, and possibly mass spectrometry to confirm identity and integrity. The final purified protein can be stored as a lyophilized powder and reconstituted in deionized sterile water to concentrations of 0.1-1.0 mg/mL .

How should researchers optimize storage conditions for maximum stability of purified protein?

Based on provided specifications, researchers should follow these guidelines to maintain maximum stability of Recombinant S. typhimurium Bifunctional protein aas :

The inclusion of 6% Trehalose in the storage buffer is particularly important as it acts as a protein stabilizer, preventing denaturation during freeze-thaw cycles. When reconstituting lyophilized protein, brief centrifugation prior to opening is recommended to ensure all material is at the bottom of the vial . For long-term storage of reconstituted protein, addition of glycerol (typically to a final concentration of 50%) helps prevent freeze damage.

How can Recombinant S. typhimurium Bifunctional protein aas contribute to vaccine development research?

While the specific role of Bifunctional protein aas in vaccine development is not directly addressed in the search results, insights can be drawn from research on attenuated Salmonella typhimurium vaccine vectors. Recombinant S. typhimurium strains are being developed as vaccine delivery systems using regulated delayed attenuation and lysis systems, as evidenced by research delivering infectious bronchitis virus proteins and heterologous O-antigens . In this context, Bifunctional protein aas could potentially be utilized in several ways:

• As a potential vaccine antigen:

  • If conserved across Salmonella strains and immunogenic, it might serve as a target

  • Recombinant expression facilitates testing of immune responses

• In attenuated strain development:

  • Understanding aas function could inform metabolic engineering of vaccine strains

  • Mutations in metabolic genes like aas might contribute to attenuation strategies

• As a fusion partner for heterologous antigens:

  • The protein's bifunctional nature might provide stable scaffolding for antigen display

  • Fusion proteins incorporating aas domains could potentially enhance immunogenicity

How does Bifunctional protein aas relate to Salmonella's regulated delayed attenuation systems?

Regulated delayed attenuation systems in Salmonella typhimurium, as described in the search results, utilize tightly controlled gene expression mechanisms involving the araC PBAD activator-promoter . These systems often incorporate mutations in genes like asdA and regulated expression of genes like murA to control bacterial attenuation and lysis in vivo . While Bifunctional protein aas is not specifically mentioned in these systems, understanding its potential metabolic roles could inform development of similar attenuation strategies.

The regulated delayed attenuation approach described in the research involves:

• Deletion of the sifA gene to prevent Salmonella-containing vacuole formation

• ΔasdA mutation combined with arabinose-regulated murA expression

• Controlled lysis in vivo to release plasmid DNA encoding heterologous antigens

This system has shown promise in delivering proteins like the S1 protein of infectious bronchitis virus, providing significant protection against viral challenge in chicken models . Similar approaches could potentially incorporate aas-related mechanisms if this protein is found to play roles in bacterial persistence or metabolism during infection.

What methods should researchers use to investigate Bifunctional protein aas in oxidative stress responses?

Based on information from search result about Salmonella's response to oxidative stress, researchers investigating Bifunctional protein aas in this context should consider these methodological approaches:

• Proteogenomic analysis:

  • Use Tn-seq with saturated Tn5 insertion libraries exposed to H2O2 at different concentrations (2.5 mM and 3.5 mM)

  • Apply data-dependent acquisition (DDA) proteomics to identify differentially expressed proteins

  • Implement targeted proteomics to confirm upregulation in response to oxidative stress

• Genetic validation:

  • Generate defined aas deletion mutants

  • Perform phenotypic evaluation of mutants under oxidative stress conditions

  • Conduct complementation studies with the wild-type gene

• Experimental design for H2O2 sensitivity testing:

This systematic approach would determine whether Bifunctional protein aas is among the 137 genes putatively required for H2O2 resistance in S. Typhimurium, similar to the comprehensive assessment described in the research .

How can researchers utilize Recombinant S. typhimurium Bifunctional protein aas in heterologous expression systems?

Researchers investigating heterologous expression systems can leverage approaches similar to those described for O-antigen expression in Salmonella . Based on these methodologies, potential strategies for utilizing Recombinant S. typhimurium Bifunctional protein aas include:

• Expression platform development:

  • Create Asd+ plasmid systems similar to pCZ1 described in the research

  • Incorporate the aas gene under appropriate promoter control

  • Use balanced lethal systems involving ΔasdA mutations and Asd+ plasmids

• Regulated expression strategies:

  • Implement arabinose-inducible systems (araC PBAD) for controlled expression

  • Design conditional expression systems that respond to specific environmental cues

  • Develop dual-expression systems for both homologous and heterologous proteins

• Validation approaches:

  • Perform immunoblotting to confirm expression efficiency

  • Use specific antibodies to track protein localization

  • Implement functional assays to verify activity of the expressed protein

The successful expression of heterologous O-antigens in Salmonella Typhimurium through recombinant plasmids described in search result provides a methodological framework that could be adapted for exploring aas expression in various contexts.

What role might Bifunctional protein aas play in Salmonella pathogenesis and host-pathogen interactions?

While the specific role of Bifunctional protein aas in pathogenesis is not directly addressed in the search results, its potential functions can be inferred based on Salmonella pathogenesis mechanisms described. As a bifunctional protein potentially involved in lipid metabolism, aas might contribute to:

• Membrane remodeling during infection:

  • Adaptation to intracellular environments

  • Modification of membrane composition in response to host defenses

  • Maintaining membrane integrity during exposure to antimicrobial factors

• Salmonella-containing vacuole (SCV) formation or maintenance:

  • The research mentions sifA gene involvement in SCV biogenesis

  • Membrane-associated proteins like aas could potentially contribute to this process

  • Lipid metabolism functions might support membrane dynamics during infection

• Resistance to host defense mechanisms:

  • Potential involvement in responses to oxidative stress, similar to genes identified in the H2O2 response study

  • Adaptation to nutrient limitation in host environments

  • Evasion of host immune recognition through membrane modifications

Investigation would require generating defined aas deletion mutants, conducting in vitro infection assays with various cell types, and evaluating virulence in appropriate animal models.

How does Bifunctional protein aas compare structurally and functionally across different bacterial species?

• Consist of 719 amino acids

• Can be expressed recombinantly with N-terminal His tags

• Are purified using similar methodologies

• Share similar storage and handling requirements

For a thorough comparative analysis, researchers should consider:

• Sequence alignment approaches:

  • Multiple sequence alignment of aas proteins from various bacterial species

  • Phylogenetic analysis to determine evolutionary relationships

  • Identification of conserved domains and variable regions

• Structural comparison methods:

  • Homology modeling based on crystal structures if available

  • Secondary structure prediction and comparison

  • Domain architecture analysis

• Functional conservation testing:

  • Cross-complementation studies between species

  • Biochemical assay standardization across orthologs

  • Heterologous expression of aas variants in model systems

What are the critical parameters for validating the functional activity of purified Recombinant S. typhimurium Bifunctional protein aas?

Validating the functional activity of purified Recombinant S. typhimurium Bifunctional protein aas requires assays specific to its biochemical functions. Based on its classification as a bifunctional protein, researchers should consider these validation approaches:

• Initial quality assessment:

  • SDS-PAGE to confirm >90% purity as specified

  • Western blotting with anti-His antibodies to verify identity

  • Circular dichroism to confirm proper folding

  • Dynamic light scattering to assess homogeneity

• Functional activity assays:

  • Enzymatic activity measurements based on predicted functions

  • If involved in acyl-CoA metabolism, measure ATP consumption or acyl-CoA formation

  • For phospholipid-related activities, assess membrane lipid modifications

• Structural integrity validation:

  • Thermal shift assays to assess stability and ligand binding

  • Limited proteolysis to confirm domain organization

  • Size exclusion chromatography to determine oligomeric state

• Biological function verification:

  • Complementation of aas deletion mutants

  • Assessment of phenotype restoration in appropriate model systems

  • In vitro reconstitution of relevant biochemical pathways

How should researchers approach experimental design when studying Bifunctional protein aas in the context of vaccine development?

When studying Bifunctional protein aas in vaccine development contexts, researchers should adopt experimental designs similar to those used in the Salmonella typhimurium vaccine candidate studies described in the search results . Key considerations include:

• Antigen delivery system design:

  • Construction of attenuated S. typhimurium strains with regulated delayed systems

  • Development of balanced lethal plasmid systems (similar to ΔasdA mutation with Asd+ plasmids)

  • Incorporation of regulated promoters (like araC PBAD) for controlled expression

• Expression validation protocol:

  • Verification of protein expression in appropriate cell lines (e.g., HD11 cells as used in )

  • Immunoblotting to confirm antigen production

  • Localization studies to determine cellular distribution

• Immunogenicity assessment framework:

  • Evaluation of antibody production against target antigens

  • Cytokine profiling to characterize immune response types

  • T-cell response analysis through appropriate assays

• Protection studies design:

  • Challenge models in appropriate animal systems

  • Tissue-specific protection assessment (e.g., lacrimal gland, trachea, cloaca protection rates)

  • Histopathological examination to evaluate tissue damage reduction

These methodological approaches should be adapted specifically to the hypothesized role of Bifunctional protein aas in the vaccine development context, whether as an antigen itself or as part of the delivery system.

What advanced analytical techniques are most suitable for studying interactions of Recombinant S. typhimurium Bifunctional protein aas?

Multiple analytical techniques can be employed to study the interactions of Recombinant S. typhimurium Bifunctional protein aas with other molecules. Based on its potential roles in metabolism and bacterial physiology, these techniques would be particularly valuable:

• Protein-protein interaction analysis:

  • Co-immunoprecipitation using the His-tag for pulldown

  • Bacterial two-hybrid systems to screen for interaction partners

  • Crosslinking mass spectrometry to map interaction interfaces

  • Proximity labeling approaches to identify interaction networks in vivo

• Protein-lipid interaction studies:

  • Liposome binding assays with fluorescently labeled proteins

  • Lipid overlay assays to determine specificity

  • Monolayer surface pressure measurements for membrane interactions

  • Microscale thermophoresis for quantitative binding parameters

• Structural investigation approaches:

  • X-ray crystallography of purified protein with potential ligands

  • Nuclear magnetic resonance for mapping interaction sites

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes

• Systems biology integration:

  • Interactome mapping in various environmental conditions

  • Network analysis to position aas in relevant biochemical pathways

  • Multi-omics integration to understand context-dependent functions

These techniques would provide complementary data to build a comprehensive understanding of how Bifunctional protein aas functions within bacterial systems and during host-pathogen interactions.