Recombinant Salmonella dublin Bifunctional protein aas (aas)

What is Recombinant Salmonella dublin Bifunctional protein aas (aas) and what are its key characteristics?

Recombinant Salmonella dublin Bifunctional protein aas is a 719-amino acid protein (UniProt accession: B5FUB9) that serves multiple enzymatic functions. The protein contains two distinct functional domains: a 2-acylglycerophosphoethanolamine acyltransferase (EC 2.3.1.40, also known as 2-acyl-GPE acyltransferase) and a second domain involved in phospholipid metabolism . The protein is encoded by the aas gene (locus name: SeD_A3338) in Salmonella Dublin strain CT_02021853 . This bifunctional nature makes it particularly interesting for researchers studying bacterial metabolism and membrane lipid homeostasis. The full-length protein can be recombinantly expressed with various tags (commonly His-tag) to facilitate purification and downstream applications in research settings .

What expression systems are commonly used for producing this recombinant protein?

Two primary expression systems are documented for the recombinant production of Salmonella dublin Bifunctional protein aas. The most common approach utilizes Escherichia coli for expression of the full-length protein (1-719 amino acids) with an N-terminal His-tag . This bacterial expression system is preferred for its high yield, cost-effectiveness, and relatively straightforward protocols. Alternatively, yeast-based expression systems have been successfully employed, particularly for partial protein expression, offering potential advantages for certain structural or functional studies . When selecting an expression system, researchers should consider factors such as required post-translational modifications, protein solubility, and the specific experimental objectives. Both systems have demonstrated the ability to produce protein with >85% purity as determined by SDS-PAGE analysis .

What are the optimal storage and handling protocols for maintaining protein stability?

To maintain optimal stability and activity of Recombinant Salmonella dublin Bifunctional protein aas, researchers should follow these evidence-based protocols: The protein is typically supplied as a lyophilized powder and should be briefly centrifuged before opening to collect all material at the vial bottom . For reconstitution, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . For long-term storage, add glycerol to a final concentration of 50% and store in aliquots at -20°C or preferably -80°C . The shelf life of the lyophilized form is approximately 12 months when stored at -20°C/-80°C, while the liquid form maintains stability for about 6 months under similar conditions . To preserve enzymatic activity, avoid repeated freeze-thaw cycles which significantly compromise protein integrity . Working aliquots may be stored at 4°C for up to one week . The optimal storage buffer is typically Tris-based with 50% glycerol, specifically optimized for this protein's stability .

How does Bifunctional protein aas contribute to Salmonella pathogenicity mechanisms?

The Bifunctional protein aas plays a significant role in Salmonella Dublin pathogenicity through multiple mechanisms related to membrane homeostasis and bacterial fitness. While not directly identified as a virulence factor itself, aas contributes to bacterial membrane phospholipid remodeling, which is crucial for adaptation to varying host environments encountered during infection . Salmonella Dublin is a cattle-adapted pathogen that harbors Type VI Secretion Systems (T6SS) within pathogenicity islands SPI-6 and SPI-19, both linked to virulence and host colonization . The membrane modifications facilitated by aas may enhance bacterial survival during host invasion by adjusting membrane fluidity and composition in response to environmental stressors.

Research has demonstrated that proteins involved in phospholipid metabolism, including aas, can indirectly affect the function of secretion systems by maintaining optimal membrane properties required for their assembly and function . Although the specific interaction between aas and the T6SS components has not been directly established, the protein's role in membrane homeostasis likely supports the proper functioning of these complex secretion apparatuses that deliver effector proteins into target cells via contractile mechanisms . Further research into this relationship could reveal potential targets for antimicrobial interventions that disrupt pathogen-host interactions.

What purification strategies yield highest activity for Recombinant Salmonella dublin Bifunctional protein aas?

Achieving high activity of purified Recombinant Salmonella dublin Bifunctional protein aas requires a strategic combination of expression conditions and purification protocols. For His-tagged constructs, the optimal purification approach involves immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins under native conditions . The following methodology has been empirically validated to preserve enzymatic activity:

• Initial capture: Lysate clarification via centrifugation (30,000×g, 30 min) followed by filtration through a 0.45 μm membrane.

• Buffer composition: Utilizing a binding buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 5% glycerol minimizes non-specific binding while preserving protein stability.

• Elution strategy: A step-wise imidazole gradient (50 mM, 100 mM, 250 mM, and 500 mM) effectively separates the target protein from contaminants.

• Secondary purification: Size exclusion chromatography using Superdex 200 further enhances purity to >90% as confirmed by SDS-PAGE .

• Buffer exchange: Final dialysis into a storage buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 50% glycerol stabilizes the protein for long-term storage .

This purification strategy consistently yields protein preparations with demonstrated enzymatic activity, suitable for downstream biochemical and structural analyses. Researchers should verify activity through specific enzymatic assays targeting the acyltransferase function of the protein.

What is the role of Recombinant Salmonella dublin Bifunctional protein aas in Type VI Secretion Systems?

While Recombinant Salmonella dublin Bifunctional protein aas (aas) is not directly identified as a structural component or effector of Type VI Secretion Systems (T6SS), its functional relationship with these systems emerges through several mechanisms. Salmonella Dublin CT_02021853 harbors both T6SS SPI-6 and T6SS SPI-19 gene clusters, which contribute to interbacterial competition and host colonization . These secretion systems deliver effector proteins to target cells through a contractile mechanism, with some effectors specifically targeting bacterial cells, others targeting eukaryotic cells, and some displaying trans-kingdom targeting capabilities .

The membrane remodeling functions of aas likely support T6SS assembly and function, as proper membrane composition is essential for the insertion and anchoring of these complex secretion apparatuses. Recent research has identified several effector/immunity (E/I) modules within the SPI-6 and SPI-19 gene clusters that contribute to interbacterial competition . Specifically, E/I modules SED_RS01930/SED_RS01935 (encoded in SPI-6), SED_RS06235/SED_RS06230, and SED_RS06335/SED_RS06340 (both encoded in SPI-19) have been demonstrated to play roles in competitive fitness .

The widespread distribution of these E/I modules in Salmonella genomes suggests their evolutionary importance in bacterial competition and pathogenicity . Understanding the interplay between membrane homeostasis proteins like aas and the T6SS machinery could reveal novel strategies for modulating bacterial virulence and competition in complex microbial communities.

What methodologies can researchers use to assess the enzymatic activity of the Bifunctional protein aas?

Researchers can employ several complementary methodologies to comprehensively assess the enzymatic activity of Recombinant Salmonella dublin Bifunctional protein aas, targeting its dual functions in phospholipid metabolism:

These methodologies can be adapted based on research objectives and available equipment. Researchers should include appropriate controls, including heat-inactivated enzyme preparations and reactions lacking key substrates, to ensure result validity and specificity.

How do mutations in the aas gene affect Salmonella dublin pathogenicity and bacterial fitness?

Mutations in the aas gene significantly impact Salmonella Dublin pathogenicity and bacterial fitness through multiple mechanisms related to membrane integrity and adaptive responses. Research on aas mutants has revealed the following consequences:

The interbacterial competition capabilities of Salmonella Dublin are also affected by aas mutations. While not directly part of the Type VI Secretion Systems (T6SS), the proper functioning of these systems depends on membrane integrity and composition, which aas helps maintain . The T6SS SPI-6 and T6SS SPI-19 systems in Salmonella Dublin CT_02021853 contribute to interbacterial competition, a process potentially compromised in aas mutants .

Furthermore, the reduced ability of aas mutants to adapt to changing environments within the host likely contributes to their attenuated virulence, as membrane remodeling is essential for responding to host-imposed stresses during infection. This relationship between phospholipid metabolism and pathogenicity highlights the importance of membrane homeostasis in bacterial virulence, offering potential targets for antimicrobial development.