Recombinant Salmonella agona ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Salmonella agona?

ATP synthase subunit c (atpE) is a component of the F0 sector of the F-type ATP synthase in Salmonella agona. This highly conserved membrane protein forms the c-ring of the F0 sector, which is crucial for proton translocation across the bacterial membrane during ATP synthesis. In S. agona, atpE is a 79-amino acid protein with the sequence MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . This protein plays a fundamental role in the bacterial energy metabolism, converting the electrochemical gradient across the membrane into chemical energy in the form of ATP.

How is recombinant Salmonella agona atpE protein typically produced?

Recombinant S. agona atpE protein is typically produced using E. coli expression systems. The atpE gene is cloned into an appropriate expression vector, often with an N-terminal or C-terminal tag (commonly His-tag) to facilitate purification. The expression construct is then transformed into E. coli, followed by induction of protein expression, cell lysis, and purification using affinity chromatography. For research-grade production, the purified protein is generally supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE .

The expression and purification process typically follows this workflow:

• Gene synthesis or PCR amplification of the atpE gene

• Cloning into an expression vector with appropriate tag

• Transformation into E. coli expression host

• Culture growth and protein expression induction

• Cell harvesting and lysis

• Affinity purification using the fusion tag

• Buffer exchange and concentration

• Quality control testing (purity, identity, activity)

• Lyophilization for long-term storage

What is the structural similarity between atpE proteins from different Salmonella serovars?

The atpE proteins from different Salmonella serovars show remarkably high conservation. For instance, the atpE proteins from Salmonella agona (UniProt: B5EZ01) and Salmonella paratyphi A (UniProt: B5BIP1) share identical amino acid sequences (MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA) . This high degree of conservation is not surprising given the critical role of atpE in energy metabolism and the evolutionary relationship between Salmonella serovars. This conservation has important implications for research, particularly in the development of broad-spectrum vaccines or antimicrobials targeting atpE.

How should recombinant atpE protein be reconstituted and stored for optimal stability?

For optimal stability and activity of recombinant atpE protein, follow these evidence-based reconstitution and storage protocols:

Reconstitution Protocol:

• Centrifuge the vial briefly to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended 50%)

• Prepare small aliquots to avoid repeated freeze-thaw cycles

Storage Recommendations:

• Long-term storage: -20°C or -80°C in aliquots containing glycerol

• Working aliquots: 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of activity

The protein is typically stored in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability during freeze-drying and reconstitution. Trehalose serves as a cryoprotectant and stabilizing agent for the protein structure.

What expression systems are most effective for producing functional recombinant atpE protein?

Several expression systems have been developed for producing functional recombinant atpE protein, each with distinct advantages:

E. coli expression systems are most commonly used for laboratory-scale production, typically with an N-terminal His-tag to facilitate purification . For functional studies, it's important to ensure proper membrane integration, which may require specialized membrane protein expression systems or reconstitution into liposomes post-purification.

What purification methods yield the highest purity of recombinant atpE protein?

To achieve high-purity recombinant atpE protein, a multi-step purification strategy is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

• Intermediate Purification: Ion exchange chromatography to remove proteins with similar affinity to the IMAC resin

• Polishing: Size exclusion chromatography to remove aggregates and obtain homogeneous protein

For membrane proteins like atpE, additional considerations include:

• Addition of appropriate detergents (e.g., DDM, LDAO) in purification buffers to maintain protein solubility

• Optimization of detergent concentration to avoid protein aggregation

• Consideration of amphipols or nanodiscs for stabilizing the native structure

Using this approach, purity greater than 90% can typically be achieved as determined by SDS-PAGE . For applications requiring ultra-high purity, additional chromatographic steps or alternative tagging strategies may be employed.

How can recombinant S. agona atpE be used in vaccine development strategies?

Recombinant S. agona atpE can be utilized in several vaccine development strategies:

• As a carrier protein for antigenic epitopes:
  The atpE protein can be engineered to present foreign antigenic epitopes when expressed in attenuated Salmonella vaccine strains. This approach takes advantage of the strong translation signals of the atpE gene, which has been incorporated into expression vectors designed for vaccine antigen delivery .

• For targeted secretion systems:
  The atpE gene's translation signals can be used in vectored vaccine design to enhance the expression of heterologous antigens. Specifically, vectors containing "the strong translation signals of the Escherichia coli atpE gene" have been developed to achieve selective expression of vaccine antigens within eukaryotic cells using Salmonella as carrier strains .

• In Type I secretion systems:
  Type I (ATP-binding cassette) secretion systems are particularly suited for recombinant extracellular expression systems in Salmonella due to their low complexity and terminal location of the secretion signal. These systems can be engineered to incorporate atpE-based constructs for improved secretory delivery of recombinant vaccines .

Despite these potential applications, it's important to note that "immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen" . This limitation is partly due to the lack of efficient secretory expression systems, which researchers are actively working to address.

What role does atpE play in Salmonella persistence and how can recombinant atpE be used to study this phenomenon?

The role of atpE in Salmonella persistence is an emerging area of research, particularly in the context of S. Agona transitioning from acute to persistent infection. Recent phylogenomic analysis of S. Agona isolates from UK infections (2004-2020) has revealed intriguing patterns:

• Genome structure variation: During early stages of persistent infection (3 weeks-3 months), there is an increase in both SNP variation and genome structure rearrangements, potentially reflecting a population expansion or immune evasion mechanism .

• Metabolic adaptation: ATP synthase components, including atpE, may play a crucial role in the metabolic adaptations required for persistent infection within host environments.

• Biofilm formation: S. Agona isolates from patients with convalescent and temporary carriage showed significantly poorer biofilm formation ability compared to isolates from acute illness . This suggests metabolic and regulatory changes during persistent infection that may involve ATP synthase components.

Recombinant atpE can be utilized to study these phenomena through:

• Structure-function analysis: Comparing atpE sequence and structure between isolates from acute versus persistent infections

• Protein interaction studies: Identifying potential binding partners that differ between acute and persistent phases

• Immunological investigations: Assessing how atpE recognition by the host immune system changes during infection progression

• Metabolic flux analysis: Examining how ATP synthesis efficiency changes as Salmonella transitions to persistence

How can CRISPR-based technologies be optimized for studying atpE gene function in Salmonella?

CRISPR-based technologies offer powerful approaches for studying atpE gene function in Salmonella, particularly when utilizing protein-centric CRISPR guide design tools like CRISPR-TAPE:

• Targeted mutagenesis of specific amino acid residues:
  CRISPR-TAPE allows researchers to directly query gRNAs targeting specific amino acids or positions of interest within the atpE protein sequence, rather than using traditional gene-centric approaches . This is especially valuable for targeting:

  • Functional residues in the proton channel

  • Conserved motifs across Salmonella species

  • Sites of potential post-translational modifications

• HDR-based precise editing:
  For optimal homology-directed repair (HDR) efficiency when introducing specific mutations, guide RNAs should be designed to ensure the nuclease cut site is within 30 nucleotides of the desired mutation site . CRISPR-TAPE can optimize this process for atpE modifications.

• Base editing applications:
  While CRISPR base editing using Cas9-cytidine/adenine deaminase fusions is limited to certain amino acid substitutions, CRISPR-TAPE can guide the selection of editable sites within atpE and identify where HDR approaches would be necessary for other substitutions .

• Experimental workflow optimization:
  When targeting multiple residues within atpE, CRISPR-TAPE offers significant time savings (estimated 300× faster per targeted residue) compared to traditional gene-centric gRNA design tools .

For researchers attempting to engineer atpE in Salmonella, the following CRISPR experimental approach is recommended:

• Use CRISPR-TAPE to identify optimal gRNAs targeting specific residues of interest

• Select appropriate CRISPR system (standard Cas9, base editors, or prime editors) based on desired modification

• Design appropriate repair templates for HDR approaches

• Validate edits through sequencing and functional assays specific to ATP synthase activity

What approaches can address poor solubility and aggregation of recombinant atpE protein?

ATP synthase subunit c (atpE) is a highly hydrophobic membrane protein that often poses solubility challenges. Researchers can employ these evidence-based strategies to address poor solubility and aggregation:

• Optimization of expression conditions:

  • Lower induction temperature (16-18°C)

  • Reduced IPTG concentration (0.1-0.5 mM)

  • Extended expression time (overnight)

  • Use of specialized E. coli strains (C41/C43) designed for membrane protein expression

• Detergent selection and optimization:

  Detergent	Critical Micelle Concentration	Best Applications
  DDM	0.17 mM	Initial extraction, mild
  LDAO	1-2 mM	High extraction efficiency
  Fos-Choline-12	1.5 mM	Stringent extraction, high stability
  Digitonin	0.5 mM	Gentle extraction, maintains complexes

  Detergent screening should be performed to identify optimal conditions for atpE solubilization.

• Alternative solubilization approaches:

  • Amphipols (A8-35) for detergent-free handling

  • Nanodiscs for reconstitution into membrane-like environment

  • SMALPs (styrene maleic acid lipid particles) for native lipid environment preservation

• Buffer optimization:

  • Include glycerol (10-20%) to prevent aggregation

  • Optimize salt concentration (typically 150-300 mM NaCl)

  • Test different pH conditions (typically pH 7.0-8.0)

  • Add stabilizing agents like arginine or trehalose

• Co-expression with chaperones:

  • GroEL/GroES system

  • DnaK/DnaJ/GrpE system

These approaches should be systematically tested and optimized for the specific experimental context and downstream applications.

How can researchers validate the functional integrity of purified recombinant atpE protein?

Validating the functional integrity of purified recombinant atpE protein is crucial for ensuring experimental reliability. Several complementary approaches can be employed:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure content

  • Thermal shift assays to assess protein stability

  • Size exclusion chromatography to confirm monodispersity

  • Dynamic light scattering to detect aggregation

• Membrane integration assays:

  • Reconstitution into liposomes and measurement of proton conductance

  • Proteoliposome-based ATP synthesis assays

  • Patch-clamp electrophysiology for single-channel recordings

• Binding studies:

  • Interaction with known binding partners (other ATP synthase subunits)

  • Binding to specific inhibitors (e.g., oligomycin, DCCD)

  • Surface plasmon resonance or microscale thermophoresis to quantify binding affinities

• Functional complementation:

  • Rescue of atpE-deficient bacterial strains

  • Restoration of ATP synthesis in membrane vesicles from mutant strains

• Structural studies:

  • Negative-stain electron microscopy to visualize c-ring formation

  • Cryo-EM analysis of reconstituted ATP synthase complexes

  • X-ray crystallography of purified c-subunit oligomers

A combination of these approaches provides comprehensive validation of recombinant atpE functionality, with the specific methods selected based on available equipment and experimental objectives.

What strategies can mitigate batch-to-batch variability in recombinant atpE protein production?

Batch-to-batch variability in recombinant atpE protein production can significantly impact experimental reproducibility. Implement these evidence-based strategies to minimize variability:

• Standardized seed culture preparation:

  • Use glycerol stocks from the same transformation event

  • Standardize culture density before induction (OD600 0.6-0.8)

  • Implement strict temperature and aeration controls

• Quality control benchmarks:

  Quality Parameter	Acceptance Criteria	Method
  Purity	>90%	SDS-PAGE, densitometry
  Identity	Positive confirmation	Western blot, mass spectrometry
  Concentration	Within 10% of target	Bradford/BCA assay
  Activity	>80% of reference standard	Application-specific assay
  Endotoxin	<0.1 EU/µg protein	LAL test

  Establish these parameters for each production batch.

• Process automation:

  • Automated expression systems with precise control of conditions

  • Standardized purification using FPLC systems with programmed methods

  • Robotic liquid handling for consistent buffer preparation

• Standard operating procedures (SOPs):

  • Detailed documentation of all procedures

  • Training protocols to ensure consistent technique across operators

  • Regular equipment calibration and maintenance

• Reference standards:

  • Maintain a reference standard from a well-characterized batch

  • Compare each new batch to the reference using multiple parameters

  • Implement statistical process control methods

• Storage standardization:

  • Consistent aliquot volumes and concentrations

  • Validated freeze-thaw stability data

  • Standardized reconstitution protocols

Implementing these strategies creates a robust production framework that significantly reduces batch-to-batch variability, enhancing experimental reproducibility and reliability.

How might atpE be exploited as a target for novel antimicrobial development?

ATP synthase subunit c (atpE) represents a promising target for novel antimicrobial development due to its essential role in bacterial energy metabolism and its structural conservation across bacterial species. Several research avenues are being explored:

• Small molecule inhibitors:

  • Development of compounds that bind specifically to bacterial atpE but not human ATP synthase

  • Targeting the c-ring to disrupt proton translocation

  • Structure-based drug design using the known atpE sequence (MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA)

• Peptide-based inhibitors:

  • Design of antimicrobial peptides that interact specifically with the c-subunit

  • Development of peptide mimetics that disrupt c-ring assembly

  • Targeting of exposed regions unique to bacterial atpE

• Combination therapies:

  • Exploiting synergy between atpE inhibitors and existing antibiotics

  • Dual targeting of ATP synthesis and membrane integrity

  • Development of delivery systems to enhance penetration of atpE inhibitors

• Resistance considerations:

  • Study of natural variations in atpE sequences across bacterial species

  • Identification of resistance mechanisms and compensatory mutations

  • Targeting of highly conserved regions to minimize resistance development

• Therapeutic index optimization:

  • Structural and functional differences between bacterial and mammalian ATP synthase

  • Development of selective inhibitors with minimal host toxicity

  • Bioavailability enhancement for systemic applications

The high conservation of atpE across Salmonella serovars suggests that successful antimicrobials targeting this protein could have broad activity against multiple pathogenic strains.

What insights might comparative studies of atpE across different Salmonella serovars provide?

Comparative studies of atpE across different Salmonella serovars can yield valuable insights into bacterial evolution, host adaptation, and pathogenesis:

• Evolutionary conservation and divergence:

  • The identical amino acid sequences observed between S. agona and S. paratyphi A atpE proteins (MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA) suggest strong evolutionary pressure to maintain this sequence.

  • Comparative genomics could reveal whether this conservation extends to other serovars and related enterobacteria.

• Host adaptation mechanisms:

  • Analysis of subtle variations in atpE sequences or expression patterns across serovars with different host preferences.

  • Investigation of how atpE contributes to adaptation to different host environments (e.g., human vs. animal hosts).

• Pathogenesis and persistence:

  • Correlation of atpE sequence or expression patterns with virulence traits.

  • Investigation of the role of atpE in the transition from acute to persistent infection .

  • Analysis of genomic structural variations involving the atpE gene during different stages of infection.

• Metabolic adaptations:

  • Comparison of ATP synthase efficiency across serovars with different metabolic requirements.

  • Investigation of atpE regulation in response to environmental stressors.

  • Analysis of atpE's role in biofilm formation, which has been shown to vary between acute and persistent S. Agona infections .

• Immune evasion strategies:

  • Analysis of atpE antigenicity across serovars.

  • Investigation of whether the genomic structural variations observed during persistent infection affect atpE expression or function.

These comparative studies could inform both fundamental understanding of Salmonella biology and applied aspects such as vaccine development and antimicrobial strategies.

How can advanced structural biology techniques enhance our understanding of atpE function in Salmonella?

Advanced structural biology techniques are revolutionizing our understanding of membrane proteins like atpE and can provide critical insights into its function in Salmonella:

• Cryo-Electron Microscopy (Cryo-EM):

  • High-resolution structures of the complete ATP synthase complex

  • Visualization of different conformational states during the catalytic cycle

  • Structural basis of c-ring rotation coupled to proton translocation

  • Sample preparation using the purified recombinant atpE (amino acid sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA) reconstituted into appropriate membrane mimetics

• Integrated Structural Biology Approaches:

  Technique	Resolution	Key Information Provided
  X-ray Crystallography	Atomic (1-3Å)	Precise amino acid positions, binding sites
  Cryo-EM	Near-atomic (2-4Å)	Conformational states, complex assembly
  NMR Spectroscopy	Atomic (local)	Dynamics, interactions in membrane
  Mass Spectrometry	N/A	Stoichiometry, post-translational modifications
  Molecular Dynamics	N/A	Conformational flexibility, energy landscapes

• Time-resolved techniques:

  • Time-resolved cryo-EM to capture intermediate states during ATP synthesis

  • Time-resolved spectroscopy to monitor proton translocation events

  • Correlation of structural changes with functional states

• In situ structural biology:

  • Cryo-electron tomography of Salmonella cells to visualize ATP synthase in its native environment

  • Correlative light and electron microscopy to link structural and functional observations

  • In-cell NMR to probe dynamics and interactions within the bacterial cytoplasm

• Computational approaches:

  • Molecular dynamics simulations to model atpE in membrane environments

  • Quantum mechanics/molecular mechanics (QM/MM) studies of proton translocation

  • Integrative modeling combining data from multiple experimental techniques

These advanced approaches can reveal the molecular basis of atpE function in energy conservation, potentially identifying novel targets for antimicrobial development and providing insights into the role of ATP synthase in Salmonella pathogenesis and persistence.