Recombinant Brucella suis biovar 1 ATP synthase subunit b 1 (atpF1)

What is ATP synthase subunit b 1 (atpF1) in Brucella suis and what are its key identifiers?

ATP synthase subunit b 1 (atpF1) is a component of the F0 sector of the F0F1 ATP synthase complex in Brucella suis. This membrane-embedded protein participates in the proton channel formation necessary for ATP synthesis.

Key Identifiers:

• Gene name: atpF1

• UniProt accession number: Q8G2D9

• Ordered locus names: BR0384, BS1330_I0385

• Alternative names: ATP synthase F(0) sector subunit b 1, ATPase subunit I 1, F-type ATPase subunit b 1, F-ATPase subunit b 1

What are the recommended storage conditions for recombinant atpF1?

For optimal stability and activity of recombinant Brucella suis ATP synthase subunit b 1:

• Store at -20°C for regular use

• For extended storage, maintain at -20°C or -80°C

• Avoid repeated freezing and thawing cycles

• Working aliquots may be stored at 4°C for up to one week

• The protein is typically supplied in a storage buffer containing Tris-based buffer with 50% glycerol

How is recombinant Brucella suis ATP synthase subunit b 1 produced for research applications?

Recombinant atpF1 is typically produced using heterologous expression systems:

Standard Production Method:

• Expression host: Escherichia coli (E. coli) is the common host organism for expression

• Vector construction: The atpF1 gene from Brucella suis biovar 1 (strain 1330) is cloned into an appropriate expression vector

• Expression induction: Protein expression is induced under optimized conditions

• Purification: The recombinant protein is purified using affinity chromatography or other suitable techniques

• Quality control: Purity and activity assays are performed to validate the final product

• Formulation: The purified protein is formulated in a Tris-based buffer containing 50% glycerol for stability

What experimental approaches can be used to study the role of atpF1 in Brucella pathogenesis?

Several methodological approaches have proven valuable for studying Brucella virulence factors:

• Transposon mutagenesis: Generate libraries of Brucella suis mutants using miniTn5 transposon insertion to identify virulence-associated genes

• Fluorescence-based screening: Using GFP-expressing bacteria to track intracellular survival and replication within macrophages

• Macrophage infection model: Human THP-1 cell line provides a standardized system for evaluating bacterial mutant attenuation

• Protein-protein interaction studies: Methods such as pull-down assays, bacterial two-hybrid systems, or co-immunoprecipitation to identify interaction partners

• Structure-function analysis: Site-directed mutagenesis of conserved residues to determine critical functional domains

What metabolic adaptations does Brucella suis employ for intramacrophagic survival?

Brucella suis demonstrates several key metabolic adaptations within macrophages:

Table 1: Key Metabolic Requirements for Intramacrophagic Survival

This metabolic profile suggests that Brucella suis must synthesize most of its essential building blocks within the macrophage rather than scavenging them from the host environment.

What experimental evidence exists for the role of ATP synthase in Brucella virulence?

While the search results don't specifically identify atpF1 in virulence screening studies, we can make reasoned inferences:

• Energy requirement for virulence: The intramacrophagic environment requires specialized energy production systems. In comprehensive virulence screens, 131 attenuated mutants of B. suis were detected from 10,272 Tn5 transposon mutants, with many encoding metabolic enzymes

• Metabolic adaptation: Virulence screens identified multiple metabolic pathways essential for intramacrophagic survival, suggesting that ATP production would be critically important in this environment

• Bioenergetic considerations: The F0F1 ATP synthase complex, of which atpF1 is a component, sits at the interface of proton gradient maintenance and ATP generation, processes likely essential during intracellular infection

How might structural features of atpF1 relate to its function in ATP synthesis?

The atpF1 protein structure contains specific features related to its function:

• Transmembrane regions: The sequence "LAITFGLFYLFLSRVVLPR" contains hydrophobic residues consistent with a membrane-spanning domain, crucial for anchoring in the bacterial membrane

• Oligomerization domains: Regions in the C-terminal portion likely participate in protein-protein interactions with other ATP synthase subunits

• Functional motifs: The sequence contains characteristic motifs for b-subunits of F-type ATP synthases, which typically form a peripheral stalk connecting F0 and F1 sectors

What are the challenges in studying ATP synthase components in bacterial pathogens?

Researchers face several methodological challenges:

• Membrane protein complexity: As membrane proteins, ATP synthase components present difficulties in:

  • Recombinant expression and purification

  • Structural analysis

  • Functional reconstitution

• Functional redundancy: Some bacteria possess multiple ATP synthase subunit isoforms, complicating genetic studies

• Essential function: Complete knockout of ATP synthase components may be lethal, necessitating conditional expression systems or partial disruption approaches

• Intracellular environment recreation: Studying ATP synthase function under conditions that mimic the intramacrophagic environment presents technical challenges

How can recombinant atpF1 be used in vaccine or therapeutic development?

Potential research applications include:

• Vaccine candidate screening: As a membrane-associated protein, atpF1 could be evaluated as a potential vaccine antigen component

• Antibody development: Recombinant atpF1 can be used to generate specific antibodies for:

  • Localization studies

  • Expression analysis under different conditions

  • Potential passive immunization approaches

• Drug target validation: Structural and biochemical studies using recombinant atpF1 could:

  • Identify critical functional residues

  • Screen for small molecule inhibitors

  • Evaluate species-specificity for targeted antimicrobial development

• Structure-based drug design: Purified protein could facilitate:

  • X-ray crystallography or cryo-EM studies

  • In silico docking studies

  • Fragment-based drug discovery approaches

How does atpF1 compare with homologous proteins in other bacterial species?

Comparative analysis reveals important insights:

• Conservation: ATP synthase components are generally conserved across bacterial species, reflecting their essential role in energy metabolism

• Phylogenetic significance: Specific sequence variations may correlate with adaptations to different ecological niches

• Pathogen-specific features: Comparison with non-pathogenic bacteria may reveal adaptations specific to intracellular pathogens

What future research directions would advance our understanding of atpF1 in Brucella pathogenesis?

Several promising research avenues remain:

• Conditional expression studies: Development of inducible or repressible systems to study atpF1 function during different infection stages

• In vivo dynamics: Investigation of atpF1 expression, localization, and regulation during macrophage infection

• Integration with systems biology: Connecting ATP synthase function with global metabolic networks and virulence mechanisms

• Structural biology approaches: Detailed structural characterization of the complete F0F1 ATP synthase complex from Brucella suis

• Comparative studies: Examination of atpF1 function across different Brucella species and biovars to identify species-specific adaptations

This FAQ collection provides a foundation for researchers working with recombinant Brucella suis ATP synthase subunit b 1, from basic handling considerations to advanced experimental applications. As research progresses, our understanding of this protein's role in bacterial pathogenesis will continue to evolve.