Recombinant Bacillus thuringiensis subsp. konkukian ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Bacillus thuringiensis subsp. konkukian and what is its molecular structure?

ATP synthase subunit c (atpE) in Bacillus thuringiensis subsp. konkukian is a small, hydrophobic membrane protein that forms a critical component of the F0 sector of F-type ATP synthase. It consists of 72 amino acids with the following sequence: MSLGVIAAAIAIGLSALGAGIGNGLIVSRTIEGVARQPELKGALQTIMFIGVALVEALPIIGVVIAFIVMNK . This protein is highly hydrophobic and contains transmembrane domains that anchor it within the bacterial membrane. The protein functions as part of the proton channel, playing a direct role in proton translocation across the membrane during ATP synthesis .

In ATP synthases, multiple copies of subunit c form an oligomeric c-ring structure that serves as a rotor component within the membrane-embedded F0 complex. This c-ring works in conjunction with the F1 delta and epsilon subunits to form the central stalk rotor element of the ATP synthase machinery .

How does atpE function within the ATP synthase complex?

The atpE protein functions as a key component of the F0 channel in ATP synthase, directly participating in proton translocation across the bacterial membrane. Multiple copies of atpE (typically 10-14 subunits) assemble into a cylindrical c-ring structure that forms the central rotor of the F0 complex .

During energy generation, the proton gradient created by the respiratory chain drives the rotation of this c-ring. The sequential protonation and deprotonation of conserved acidic residues (similar to Asp61 in other species) within the atpE protein couples the proton flux to the rotational movement of the c-ring . This rotation is mechanically transmitted to the F1 sector through interaction with the gamma and epsilon subunits, ultimately driving ATP synthesis in the catalytic domain of F1 .

The functional mechanism involves:

• Proton binding to an acidic residue in the c-subunit on one side of the membrane

• Rotation of the c-ring, transporting the proton through the membrane

• Deprotonation of the residue on the opposite side of the membrane

• Transmission of the rotational energy to the F1 sector, catalyzing ATP synthesis

This proton-pumping process is essential for energy conversion in the bacterial cell, making atpE vital for cellular bioenergetics .

What are the optimal methods for cloning and expressing the atpE gene from Bacillus thuringiensis subsp. konkukian?

Based on established protocols for similar bacterial proteins, the following methodology is recommended for successful cloning and expression of atpE from B. thuringiensis subsp. konkukian:

Cloning Strategy:

• PCR amplification of the atpE gene (GenBank: BT9727_4993) using high-fidelity DNA polymerase with primers containing appropriate restriction sites (preferably NdeI/SalI based on successful approaches with similar proteins) .

• Purification of the PCR product followed by digestion with restriction enzymes.

• Ligation into an expression vector (e.g., pET-32(+)) that provides a fusion tag to aid in purification .

• Transformation into a cloning strain such as E. coli JM109 for plasmid maintenance and verification .

• Sequence confirmation to ensure no mutations were introduced during PCR.

Expression Protocol:

• Transform the verified recombinant plasmid into an expression host like E. coli BL21(DE3).

• Culture the transformed bacteria in LB medium supplemented with appropriate antibiotics.

• Induce protein expression with 0.5-1 mM IPTG when the culture reaches appropriate density (OD600 ~0.6-0.8).

• Optimize expression conditions (temperature, duration) - typically 16-20°C for 16-20 hours works well for membrane proteins to prevent inclusion body formation .

This approach has been successful for expressing similar proteins from the Bacillus genus and other bacterial species, including recombinant ATP-binding proteins described in the literature .

What purification strategies are most effective for recombinant atpE protein?

The purification of recombinant atpE protein from Bacillus thuringiensis subsp. konkukian requires careful consideration of its hydrophobic nature as a membrane protein. Based on successful approaches with similar proteins, the following strategy is recommended:

Purification Protocol:

• Cell Lysis: Harvest induced cells and lyse using a combination of enzymatic (lysozyme) and mechanical (sonication) methods in a buffer containing detergent (typically 1% n-dodecyl β-D-maltoside) to solubilize the membrane protein.

• Initial Purification: If expressed with a His-tag, use Ni-NTA affinity chromatography. The protocol should include:

  • Equilibration of Ni-NTA resin with binding buffer containing detergent

  • Incubation of cleared lysate with the resin (typically 1-2 hours at 4°C)

  • Washing with increasing concentrations of imidazole (10-30 mM) to remove non-specific binding

  • Elution with high imidazole concentration (250-300 mM)

• Further Purification: Size exclusion chromatography is effective for obtaining highly pure protein and removing aggregates.

• Detergent Exchange: If needed for functional studies, the detergent can be exchanged during size exclusion chromatography.

The purity of the protein should be verified using SDS-PAGE, which typically shows a band at approximately 8-9 kDa for the monomeric atpE protein, though this may appear larger (around 10-12 kDa) due to the presence of the His-tag .

Storage Considerations:
The purified protein is best stored in a Tris-based buffer with 50% glycerol at -20°C for short-term storage or -80°C for long-term storage. Repeated freeze-thaw cycles should be avoided, and working aliquots can be kept at 4°C for up to one week .

How can researchers assess the functionality of recombinant atpE protein?

Assessing the functionality of recombinant atpE protein is crucial for confirming that the expressed protein retains its native structural and functional properties. Several complementary approaches can be employed:

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy: To verify the secondary structure content, particularly the α-helical content characteristic of atpE proteins.

• Size Exclusion Chromatography: To determine if the protein forms the expected oligomeric state.

• Blue Native PAGE: To assess the assembly of atpE into higher-order complexes.

Functional Assays:

• Reconstitution into Liposomes: Incorporate the purified atpE into artificial liposomes and measure proton translocation using pH-sensitive fluorescent dyes.

• ATP Synthesis Assay: If combined with other ATP synthase components, measure ATP production when exposed to a proton gradient.

• Binding Studies: Assess the interaction of atpE with other ATP synthase subunits using techniques such as:

  • Far-western blotting

  • ELISA

  • Surface Plasmon Resonance (SPR)

  These methods have been successfully applied to similar proteins to confirm their binding interactions .

• Thermal Stability Assay: Methods like differential scanning fluorimetry (DSF) can be used to assess protein stability and proper folding.

Complementation Studies:
For ultimate functional validation, genetic complementation in bacterial strains with deleted or non-functional atpE genes can demonstrate if the recombinant protein retains full physiological activity.

These methodologies collectively provide a comprehensive assessment of whether the recombinant atpE protein is structurally intact and functionally active.

How can atpE be used as a molecular target for detection and quantification of bacteria?

The atpE gene offers significant potential as a molecular target for specific detection and quantification of bacteria, particularly for members of specific genera such as Mycobacterium. Research has demonstrated several advantages of targeting atpE:

Advantages of atpE as a Detection Target:

• High Specificity: The atpE gene contains regions that are highly conserved within specific bacterial genera while showing significant divergence from other genera. This allows for highly specific detection systems .

• Single Copy Gene: Unlike ribosomal genes that may have multiple copies in the genome, atpE is typically present as a single copy, enabling more accurate quantification of bacterial load .

• Essential Function: As part of the ATP synthase complex, atpE serves an essential function in bacterial energy metabolism, reducing the likelihood of gene loss and therefore providing a reliable detection target .

Methodology for Developing atpE-Based Detection Systems:

• Primer and Probe Design:

  • Conduct comprehensive sequence alignments of atpE genes from target and non-target bacteria

  • Identify regions of high conservation within the target group and divergence from non-targets

  • Design primers and probes using specialized software such as Thermo Fisher Scientific's oligo primer design tools

  • Test primer specificity against a panel of reference strains

• Optimization of PCR Conditions:

  • Determine optimal annealing temperatures, Mg²⁺ concentrations, and cycling parameters

  • For increased sensitivity, real-time PCR with specific probes is recommended

• Validation:

  • Test against clinical or environmental samples with known bacterial content

  • Compare with established detection methods

  • Determine limits of detection and quantification

Studies have successfully employed atpE-targeted PCR for the detection of Mycobacterium species in environmental samples with high specificity (100%) and sensitivity (61.54%) . This approach has proven particularly valuable for detecting bacteria in complex samples like sputum, where traditional culture methods may be time-consuming or inadequate .

What strategies can be employed to enhance the stability of recombinant atpE protein while maintaining its function?

Enhancing the stability of recombinant proteins while preserving their function is a critical challenge in protein engineering. For atpE from Bacillus thuringiensis subsp. konkukian, several evidence-based approaches can be employed:

Structure-Based Design Approach:

• Computational Prediction of Stabilizing Mutations:

  • Use molecular dynamics simulations and computational algorithms to identify potentially stabilizing mutations

  • Focus on surface residues that don't affect the functional core of the protein

  • Analyze conserved vs. variable regions across homologous proteins

• Combinatorial Library Generation:

  • Create libraries of mutants with various combinations of predicted stabilizing mutations

  • Express these variants in a suitable host system (e.g., E. coli BL21)

  • Screen for both stability and function

This approach has been successfully demonstrated with other proteins from B. thuringiensis konkukian, resulting in significantly enhanced thermal stability without compromising catalytic efficiency .

Experimental Stabilization Strategies:

• Directed Evolution:

  • Subject the atpE gene to random mutagenesis

  • Screen for variants with improved stability using thermal challenge assays

  • Select mutants that maintain function after heat treatment

• Fusion Partners and Tags:

  • Express atpE with fusion partners known to enhance stability (e.g., thioredoxin)

  • Design constructs with removable tags if they interfere with function

• Formulation Optimization:

  • Test various buffer compositions and additives (glycerol, specific ions)

  • Optimize pH and ionic strength for maximum stability

  • Consider the addition of lipids or detergents to mimic the native membrane environment

Case Study Evidence:
Research with another enzyme from B. thuringiensis konkukian (3-dehydroshikimate dehydratase) demonstrated that a triple mutant identified through structure-based design showed more than 10-fold increase in half-life at 37°C (from 15 min to 169 min) while maintaining nearly identical catalytic efficiency . This suggests that similar approaches could be effective for stabilizing atpE.

For validation of successful stabilization, researchers should measure:

• Thermal stability (half-life at various temperatures)

• Functional assays before and after thermal challenge

• Structural integrity using CD spectroscopy or other biophysical methods

• Expression yields in the chosen host system

These comprehensive approaches can significantly enhance protein stability while preserving the essential function of recombinant atpE.

How does the sequence conservation of atpE across bacterial species inform evolutionary studies and drug development?

The atpE gene exhibits notable patterns of conservation and variation across bacterial species, providing valuable insights for both evolutionary studies and therapeutic development. Analysis of sequence data reveals:

Evolutionary Implications:

Table 1: Conservation Analysis of Key Functional Residues in atpE Across Selected Bacterial Species

Implications for Drug Development:

• Target Identification:

  • The high conservation of functional regions in atpE makes it an attractive target for broad-spectrum antimicrobials

  • Studies on diarylquinoline drugs targeting mycobacterial atpE have demonstrated the therapeutic potential of this approach

• Resistance Mechanisms:

  • Research shows that specific mutations in atpE (e.g., A63P, I66M in M. tuberculosis) can confer resistance to drugs targeting this protein

  • Natural resistance in some species (e.g., M. xenopi) correlates with specific amino acid substitutions in otherwise highly conserved positions

• Species Selectivity:

  • Despite high conservation, subtle sequence differences between bacterial groups can be exploited for selective targeting

  • Understanding these differences is crucial for developing drugs with minimal impact on beneficial microbiota

The collective analysis of atpE sequence conservation provides a foundation for understanding bacterial evolution and developing targeted antimicrobial agents. The natural variants observed in resistant species can guide rational drug design to circumvent resistance mechanisms while maintaining efficacy against susceptible pathogens .

What are the key challenges in expressing and purifying functional recombinant atpE, and how can they be addressed?

The expression and purification of functional membrane proteins like atpE present several significant challenges. Based on research experience with similar proteins, the following obstacles and solutions are relevant:

Challenge 1: Low Expression Levels
Membrane proteins often express poorly in heterologous systems due to toxicity, improper folding, or aggregation.

Solutions:

• Optimization of Expression Systems:

  • Use specialized E. coli strains designed for membrane protein expression (C41(DE3), C43(DE3))

  • Consider cell-free expression systems that can accommodate membrane proteins

  • Test expression in different host organisms (yeast, insect cells) if bacterial systems prove inadequate

• Codon Optimization:

  • Analyze the codon usage pattern of the atpE gene and optimize for the expression host

  • This approach has significantly improved expression levels for other membrane proteins

• Fusion Tags and Partners:

  • Expression with fusion partners known to enhance solubility (MBP, SUMO, thioredoxin)

  • Include purification tags (His, Strep) at positions that don't interfere with protein folding

Challenge 2: Protein Misfolding and Aggregation

Solutions:

• Temperature Modulation:

  • Lower expression temperature (16-20°C) to slow protein synthesis and allow proper folding

  • Use heat shock proteins or molecular chaperones as co-expression partners

• Detergent Screening:

  • Systematic testing of different detergents for extraction efficiency and protein stability

  • Consider mild detergents like DDM, LMNG, or Brij-35 for initial extraction

• Membrane Scaffold Proteins:

  • Co-expression with membrane scaffold proteins that facilitate proper membrane insertion

Challenge 3: Maintaining Functional State During Purification

Solutions:

• Lipid Supplementation:

  • Addition of specific lipids during purification to maintain the native environment

  • Consider nanodiscs or lipid bilayer mimetics for final protein preparation

• Buffer Optimization:

  • Screen various buffer conditions (pH, salt concentration, additives)

  • Include stabilizing agents such as glycerol (as used successfully with recombinant B. thuringiensis proteins)

• Gentle Purification Methods:

  • Use flow rates and pressure conditions that minimize protein denaturation

  • Consider alternatives to traditional chromatography (e.g., affinity precipitation)

Challenge 4: Functional Validation
Confirming that the purified protein retains its native activity is particularly challenging for membrane proteins like atpE.

Solutions:

• Liposome Reconstitution:

  • Incorporate purified atpE into liposomes to restore its native membrane environment

  • Measure proton translocation using pH-sensitive fluorescent dyes

• Complex Reconstitution:

  • Attempt reconstitution with other ATP synthase components to measure ATP synthesis activity

  • Use isolated membrane fragments from atpE-deficient strains as a platform for complementation

These methodological approaches have proven successful with similar challenging membrane proteins and provide a framework for obtaining functional recombinant atpE protein.

How can researchers investigate the interaction between atpE and other components of the ATP synthase complex?

Investigating the interactions between atpE and other components of the ATP synthase complex is crucial for understanding the assembly and function of this essential enzyme. Several complementary approaches can be employed:

Biochemical Interaction Studies:

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies specific to atpE or use tagged versions of the protein

  • Pull down atpE and identify interacting partners by mass spectrometry

  • Conduct reciprocal Co-IPs using antibodies against other ATP synthase subunits

• Far-Western Blotting:

  • Separate ATP synthase components by SDS-PAGE and transfer to a membrane

  • Probe with biotinylated recombinant atpE

  • Detect binding using streptavidin-conjugated reporter systems

  This approach has been successfully used to study protein-protein interactions in similar systems .

• ELISA-Based Binding Assays:

  • Immobilize purified atpE or other ATP synthase components on plates

  • Add potential binding partners at varying concentrations

  • Detect binding using specific antibodies

  Similar approaches have shown that increasing concentrations of interacting proteins result in greater binding until saturation is reached .

Structural and Biophysical Approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Purify intact ATP synthase complexes

  • Visualize the structural arrangement of subunits including atpE

  • Compare wild-type structures with those containing modified atpE

• Cross-linking Mass Spectrometry:

  • Use chemical cross-linkers to capture transient interactions

  • Digest cross-linked complexes and identify interaction sites by mass spectrometry

  • Map the interaction interfaces between atpE and other subunits

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize atpE on sensor chips/tips

  • Measure real-time binding kinetics with other ATP synthase components

  • Determine affinity constants (KD) for different interactions

Genetic and Functional Approaches:

• Site-Directed Mutagenesis:

  • Introduce specific mutations in predicted interaction interfaces of atpE

  • Assess the impact on complex assembly and function

  • Identify critical residues for protein-protein interactions

• Bacterial Two-Hybrid System:

  • Construct fusion proteins of atpE and potential interacting partners

  • Screen for positive interactions in bacterial reporter systems

  • Verify interactions with biochemical methods

• Suppressor Mutation Analysis:

  • Identify mutations in atpE that disrupt ATP synthase function

  • Screen for second-site suppressor mutations in other subunits

  • Map functional interaction networks

These multifaceted approaches provide comprehensive insights into how atpE interacts with other ATP synthase components, contributing to our understanding of complex assembly, stability, and function in Bacillus thuringiensis subsp. konkukian.

What are the methodological considerations for using atpE as a target in bacterial detection and identification systems?

When developing atpE-based detection and identification systems for bacteria, several critical methodological considerations must be addressed to ensure specificity, sensitivity, and reliability:

Primer and Probe Design Considerations:

• Sequence Alignment and Analysis:

  • Perform comprehensive alignments of atpE sequences from target and non-target bacteria

  • Identify regions that are conserved within the target group but differ from non-targets

  • For species-specific detection, focus on regions with unique sequences to the target organism

• Primer Characteristics for Optimal Performance:

  • Design primers with the following properties:

    • Length between 18-24 nucleotides

    • GC content between 40-60%

    • Melting temperature around 54-60°C

    • Avoid runs of identical nucleotides

    • Preferably end with G or C at the 3' end to promote stable binding

  • These characteristics have been shown to significantly impact detection sensitivity

• Probe Design for Real-time PCR:

  • For TaqMan probes, design 15-30 nucleotide sequences with higher Tm than primers

  • Position probes to avoid secondary structures in the target sequence

  • Include appropriate fluorophores and quenchers for optimal signal generation

Validation Requirements:

• Analytical Specificity Testing:

  • Test primers against a panel of closely related species and common environmental bacteria

  • Include both closely related species and those from diverse phylogenetic backgrounds

  • For atpE-based detection, inclusion of various Bacillus species is particularly important

• Sensitivity Determination:

  • Establish limits of detection using serial dilutions of purified DNA

  • Compare with established gene targets (e.g., 16S rRNA, rpoB)

  • Validate with spiked samples to account for matrix effects

• Reproducibility Assessment:

  • Test inter-laboratory variation using standardized protocols

  • Evaluate intra-assay and inter-assay variation coefficients

  • Determine robustness across different PCR instruments and reagent lots

Optimization for Sample Types:

• DNA Extraction Methods:

  • Compare different extraction methods for various sample types

  • For environmental or clinical samples, evaluate both commercial kits and cost-effective methods

  • Studies have shown significant differences between chemical-based extraction and boiling methods for detection of bacterial DNA

• PCR Inhibitor Management:

  • Include internal amplification controls to detect PCR inhibition

  • Consider sample-specific purification steps to remove inhibitors

  • Evaluate the need for BSA or other PCR facilitators for complex samples

• Multiplexing Potential:

  • Assess compatibility with other gene targets for multiplex detection

  • Design primers with compatible annealing temperatures

  • Evaluate for potential cross-reactivity or primer-dimer formation

Performance Metrics:
Based on published research, atpE-based detection systems can achieve high specificity (up to 100%) with moderate to high sensitivity (61.54% in some studies) . These metrics indicate the potential of atpE as a viable target for bacterial detection systems, particularly when high specificity is required.

What emerging technologies could enhance our understanding of atpE structure and function in Bacillus thuringiensis?

Several cutting-edge technologies are poised to significantly advance our understanding of atpE structure, function, and interactions in Bacillus thuringiensis subsp. konkukian:

Advanced Structural Biology Approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Recent advances in cryo-EM have revolutionized membrane protein structural biology

  • Application to the ATP synthase c-ring could reveal detailed molecular arrangements of atpE subunits

  • Single-particle analysis could capture different conformational states during the catalytic cycle

• Integrative Structural Biology:

  • Combining multiple methodologies (X-ray crystallography, NMR, SAXS, computational modeling)

  • This approach can overcome limitations of individual techniques for membrane proteins like atpE

  • Particularly valuable for understanding dynamic aspects of atpE function

• Solid-State NMR Spectroscopy:

  • Specialized for membrane proteins in their native-like lipid environments

  • Can provide atomic-level insights into the structure and dynamics of atpE in membranes

  • Particular value for understanding proton translocation mechanisms

Functional Genomics and Systems Biology:

• CRISPR-Cas9 Genomic Engineering:

  • Precise genome editing to create subtle mutations in atpE

  • Analysis of fitness effects under various growth conditions

  • Creation of conditional knockdowns to study essentiality in different contexts

• Transcriptome and Proteome Integration:

  • Multi-omics approaches to understand atpE regulation within the ATP synthase operon

  • Identification of co-regulated genes that might functionally interact with atpE

  • Insights into adaptation of energy metabolism under different environmental stresses

Emerging Biophysical Techniques:

• Single-Molecule FRET:

  • Direct observation of conformational changes in atpE during proton translocation

  • Real-time measurement of c-ring rotation within reconstituted systems

  • Correlation of structural dynamics with ATP synthesis activity

• High-Speed Atomic Force Microscopy (HS-AFM):

  • Visualization of ATP synthase dynamics at the nanoscale

  • Direct observation of c-ring rotation in native-like membrane environments

  • Integration with electrical recordings for structure-function correlations

• Nanodiscs and Lipid Cubic Phase Technologies:

  • Improved membrane mimetics for functional reconstitution of atpE

  • Better preservation of native lipid interactions important for function

  • Enhanced stability for long-term biophysical studies

Computational Approaches:

• Enhanced Molecular Dynamics Simulations:

  • All-atom simulations of the c-ring in explicit membrane environments

  • Elucidation of proton translocation pathways and energy coupling mechanisms

  • Prediction of effects of mutations on structure and function

• Machine Learning Applications:

  • Prediction of functional residues from evolutionary sequence analysis

  • Identification of potential binding sites for small molecules

  • Design of improved recombinant atpE variants with enhanced properties

These emerging technologies, especially when applied in combination, promise to significantly expand our understanding of atpE structure and function in Bacillus thuringiensis, with broader implications for bacterial bioenergetics and potential applications in biotechnology and antimicrobial development.

How might research on atpE contribute to the development of novel antimicrobial agents?

Research on ATP synthase subunit c (atpE) holds significant promise for antimicrobial drug development, offering several unique advantages as a therapeutic target:

Strategic Advantages of atpE as an Antimicrobial Target:

• Essential Function:

  • ATP synthase is critical for energy metabolism in virtually all bacteria

  • Inhibition of atpE function directly impacts bacterial viability

  • The essentiality of this target reduces the likelihood of target-based resistance

• Structural Conservation with Selective Differences:

  • The atpE protein contains highly conserved functional domains, making it a broad-spectrum target

  • Subtle but important sequence differences exist between bacterial species that can be exploited for selectivity

  • Critical differences between bacterial and human ATP synthase can be leveraged for therapeutic selectivity

• Established Precedent:

  • The diarylquinoline class of drugs (e.g., R207910) successfully targets mycobacterial atpE

  • Specific mutations (A63P, I66M) in atpE confer resistance to these compounds, confirming the mechanism of action

  • These findings validate atpE as a druggable target in bacteria

Research Approaches for Antimicrobial Development:

• Structure-Based Drug Design:

  • Determination of high-resolution structures of Bacillus thuringiensis atpE

  • In silico screening of compound libraries against defined binding pockets

  • Rational design of inhibitors that exploit species-specific differences

• Resistance Mechanism Studies:

  • Analysis of natural resistance in species like M. xenopi, which has a Met substitution at the highly conserved Ala63 position

  • Prediction of potential resistance mutations to guide pre-emptive drug design

  • Development of dual-targeting or combination approaches to minimize resistance emergence

• Novel Inhibitor Classes:

  • Exploration beyond diarylquinolines to novel chemical scaffolds

  • Investigation of natural products that may target atpE

  • Development of peptide-based inhibitors that disrupt c-ring assembly

Potential Development Pathway:

• Target Validation:

  • Genetic and biochemical confirmation of atpE essentiality in B. thuringiensis

  • Demonstration of growth inhibition through specific atpE targeting

• Assay Development:

  • Establishment of high-throughput screening systems for atpE inhibitors

  • Development of functional assays for ATP synthase activity in membrane vesicles

  • Creation of whole-cell reporter systems to monitor ATP synthesis inhibition

• Lead Optimization:

  • Medicinal chemistry to improve potency, selectivity, and pharmacokinetic properties

  • Testing against panels of relevant bacterial species to establish spectrum of activity

  • Evaluation against mutant strains to assess resistance barriers

• Therapeutic Potential Assessment:

  • In vitro and in vivo efficacy studies in relevant infection models

  • Toxicity evaluation focusing on mitochondrial effects

  • Resistance frequency determination and mechanism characterization

The unique position of atpE in bacterial energy metabolism, combined with the emerging structural and functional data, positions this protein as a promising target for next-generation antimicrobials that could address the growing challenge of drug-resistant bacterial infections.

What is the recommended protocol for expressing and purifying recombinant Bacillus thuringiensis subsp. konkukian atpE protein?

The following detailed protocol is recommended for the successful expression and purification of recombinant Bacillus thuringiensis subsp. konkukian ATP synthase subunit c (atpE), based on established methods for similar membrane proteins:

Expression Protocol:

Materials Required:

• pET expression vector (preferably pET-32(+) with N-terminal His-tag)

• E. coli BL21(DE3) competent cells

• LB broth and agar plates with appropriate antibiotics

• IPTG (isopropyl β-D-1-thiogalactopyranoside)

• Culture incubator with shaking capability

Procedure:

• Cloning and Transformation:

  • Clone the atpE gene (GenBank: BT9727_4993) into pET-32(+) vector between NdeI and SalI restriction sites

  • Transform the construct into E. coli BL21(DE3) cells

  • Plate on LB agar containing ampicillin (100 μg/mL)

  • Incubate overnight at 37°C

• Starter Culture:

  • Inoculate a single colony into 10 mL LB broth with ampicillin

  • Incubate overnight at 37°C with shaking at 200 rpm

• Expression Culture:

  • Dilute the starter culture 1:100 into 1 L of fresh LB with ampicillin

  • Grow at 37°C with shaking until OD600 reaches 0.6-0.8

  • Cool the culture to 18°C

  • Induce protein expression with 0.5 mM IPTG

  • Continue incubation at 18°C for 16-20 hours

• Cell Harvest:

  • Centrifuge the culture at 5,000 × g for 15 minutes at 4°C

  • Wash cell pellet once with ice-cold PBS

  • Store pellet at -80°C or proceed directly to purification

Purification Protocol:

Materials Required:

• Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1% n-dodecyl β-D-maltoside (DDM), protease inhibitor cocktail

• Wash buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 0.1% DDM

• Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole, 0.05% DDM

• Ni-NTA agarose resin

• Size exclusion chromatography column (e.g., Superdex 200)

• SEC buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% DDM

• Storage buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% DDM, 50% glycerol

Procedure:

• Cell Lysis:

  • Resuspend cell pellet in lysis buffer (10 mL per gram of cells)

  • Incubate with lysozyme (1 mg/mL) for 30 minutes on ice

  • Sonicate on ice (6 cycles of 30 seconds on, 30 seconds off)

  • Centrifuge at 20,000 × g for 30 minutes at 4°C to remove cell debris

  • Ultracentrifuge the supernatant at 100,000 × g for 1 hour at 4°C

  • Collect the supernatant containing solubilized membrane proteins

• Ni-NTA Affinity Chromatography:

  • Equilibrate Ni-NTA resin with lysis buffer

  • Incubate the clarified lysate with pre-equilibrated Ni-NTA resin for 2 hours at 4°C with gentle rotation

  • Pack the resin into a column and wash with 10 column volumes of wash buffer

  • Elute bound protein with 5 column volumes of elution buffer, collecting 1 mL fractions

  • Analyze fractions by SDS-PAGE

• Size Exclusion Chromatography:

  • Pool protein-containing fractions from Ni-NTA purification

  • Concentrate to 2-5 mL using a centrifugal concentrator (10 kDa cutoff)

  • Load onto a pre-equilibrated Superdex 200 column

  • Elute with SEC buffer at a flow rate of 0.5 mL/min

  • Collect fractions and analyze by SDS-PAGE

• Storage:

  • Pool pure protein fractions and concentrate to 1-5 mg/mL

  • Add glycerol to a final concentration of 50%

  • Aliquot and flash-freeze in liquid nitrogen

  • Store at -80°C for long-term storage

Expected Results:

• The purified atpE protein should appear as a band at approximately 8-9 kDa on SDS-PAGE, potentially migrating slightly higher due to the His-tag

• Typical yield: 1-3 mg of purified protein per liter of culture

• Purity: >90% as assessed by SDS-PAGE

Quality Control:

• Verify protein identity by mass spectrometry or western blotting

• Assess oligomeric state by native PAGE or analytical SEC

• Confirm proper folding by circular dichroism spectroscopy

This protocol is optimized for the expression and purification of recombinant Bacillus thuringiensis subsp. konkukian atpE protein based on successful approaches with similar proteins .

How can researchers design and validate primers for atpE-based detection of bacteria in environmental or clinical samples?

A comprehensive approach for designing and validating primers for atpE-based bacterial detection involves multiple steps to ensure specificity, sensitivity, and reliability. The following protocol outlines the process based on successful approaches in published research:

Primer Design Protocol:

Materials and Software:

• Sequence databases (NCBI GenBank, Ensembl Bacteria)

• Sequence alignment software (MUSCLE, Clustal Omega)

• Primer design software (Primer3, Thermo Fisher Scientific Oligo Primer Design Tool)

• Oligonucleotide property analysis tools (OligoAnalyzer)

• PCR simulation software (in silico PCR)

Procedure:

• Sequence Collection and Alignment:

  • Retrieve atpE gene sequences from target bacteria and closely related non-target species

  • For B. thuringiensis subsp. konkukian, include sequences from other Bacillus species, particularly B. cereus group members

  • Perform multiple sequence alignment to identify:
    a) Conserved regions within target species/group
    b) Regions with variation between target and non-target organisms

• Target Region Selection:

  • Identify regions of 200-500 bp that show appropriate conservation patterns

  • Analyze GC content and secondary structure potential in these regions

  • Evaluate uniqueness using BLAST against comprehensive databases

• Primer Design Parameters:

  • Design primers with the following characteristics:
    a) Length: 18-24 nucleotides
    b) GC content: 40-60%
    c) Melting temperature (Tm): 54-60°C with minimal difference between forward and reverse primers
    d) Avoid runs of >3 identical nucleotides, especially G or C
    e) Ensure the 3' end of primers terminates with G or C for stronger binding
    f) Avoid regions prone to secondary structure formation

• Probe Design (for real-time PCR):

  • Design probes 15-30 nucleotides in length

  • Ensure probe Tm is 5-10°C higher than primer Tm

  • Select appropriate fluorophore and quencher combinations

  • Position the probe to avoid regions with predicted secondary structure

• In Silico Validation:

  • Perform electronic PCR against comprehensive genome databases

  • Check for potential non-specific amplification

  • Evaluate primer-dimer and hairpin formation potential

  • Adjust design as needed based on in silico results

Experimental Validation Protocol:

Materials:

• Designed primers and probes

• DNA from target and non-target bacterial species

• PCR reagents (polymerase, buffer, dNTPs)

• Real-time PCR system (for qPCR validation)

• Gel electrophoresis equipment

• DNA sequencing capability

Procedure:

• Initial PCR Optimization:

  • Test primers using gradient PCR to determine optimal annealing temperature

  • Optimize Mg²⁺ concentration and PCR cycling parameters

  • Analyze products by gel electrophoresis to confirm expected amplicon size

• Specificity Testing:

  • Test primers against DNA from:
    a) Target organism (B. thuringiensis subsp. konkukian)
    b) Closely related species (other B. thuringiensis strains, B. cereus)
    c) Distantly related bacteria (diverse panel including gram-positive and gram-negative species)

  • Sequence amplicons from target species to confirm correct target amplification

• Sensitivity Evaluation:

  • Prepare serial dilutions of target DNA (10-fold dilutions)

  • Determine limit of detection using conventional and real-time PCR

  • Establish standard curves for quantification (for qPCR)

  • Calculate PCR efficiency using the formula: E = 10^(-1/slope) - 1

• Validation with Complex Samples:

  • Spike known quantities of target bacteria into relevant matrices (environmental samples, clinical specimens)

  • Extract DNA using appropriate methods (commercial kits vs. boiling method)

  • Test primer performance in the presence of potential PCR inhibitors

  • Compare different DNA extraction methods for sensitivity and reliability

• Reproducibility Assessment:

  • Perform replicate reactions (minimum triplicate) to assess intra-assay variation

  • Repeat experiments on different days to evaluate inter-assay variation

  • If possible, test in different laboratories to assess robustness

Performance Metrics and Reporting:

• Calculate sensitivity, specificity, positive predictive value, and negative predictive value

• Report limits of detection and quantification in relevant units (copies/reaction, CFU/mL)

• Document optimal reaction conditions for future reference

• Specify any limitations observed during validation

Research has shown that properly designed and validated atpE-targeted primers can achieve high specificity (100%) with good sensitivity (61.54%) , making them valuable tools for bacterial detection in various applications.

What are common problems encountered when working with recombinant atpE protein, and how can they be resolved?

Working with recombinant ATP synthase subunit c (atpE) from Bacillus thuringiensis subsp. konkukian presents several technical challenges due to its nature as a small, hydrophobic membrane protein. The following troubleshooting guide addresses common issues and provides evidence-based solutions:

Problem 1: Low Expression Yield

Symptoms:

• Minimal or undetectable protein band on SDS-PAGE

• Low protein concentration after purification attempts

Possible Causes and Solutions:

• Toxicity to Expression Host:

  • Solution: Use specialized strains designed for toxic/membrane proteins (C41/C43)

  • Solution: Reduce induction temperature to 16-18°C and use lower IPTG concentrations (0.1-0.2 mM)

  • Solution: Use a tightly controlled expression system with minimal leaky expression

• Codon Usage Bias:

  • Solution: Analyze the atpE sequence for rare codons in the expression host

  • Solution: Either optimize the gene sequence or use strains with additional tRNAs for rare codons

• Protein Degradation:

  • Solution: Include protease inhibitors during all purification steps

  • Solution: Consider co-expression with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Solution: Reduce expression time and harvest cells earlier

Problem 2: Protein Insolubility and Aggregation

Symptoms:

• Target protein found predominantly in the insoluble fraction

• Protein precipitates during or after purification

Possible Causes and Solutions:

• Ineffective Membrane Solubilization:

  • Solution: Screen multiple detergents systematically (DDM, LMNG, Brij-35, CHAPS)

  • Solution: Optimize detergent concentration and solubilization time

  • Solution: Consider using detergent mixtures for improved extraction

• Improper Folding:

  • Solution: Reduce expression rate by lowering temperature and inducer concentration

  • Solution: Include stabilizing additives (glycerol, specific lipids, salt)

  • Solution: Test fusion partners known to enhance membrane protein folding

• Buffer Incompatibility:

  • Solution: Screen different buffer systems (Tris, HEPES, phosphate) at various pH values

  • Solution: Test different ionic strengths (100-500 mM NaCl)

  • Solution: Add stabilizing agents such as glycerol (5-50%)

Problem 3: Loss of Protein During Purification

Symptoms:

• Significant decrease in protein amount between purification steps

• Protein detected in flow-through or wash fractions

Possible Causes and Solutions:

• Poor Binding to Affinity Resin:

  • Solution: Verify tag accessibility; consider moving tag to the opposite terminus

  • Solution: Optimize binding conditions (time, temperature, buffer composition)

  • Solution: Test different affinity resins or binding buffers

• Tag Cleavage During Expression/Purification:

  • Solution: Use protease inhibitors throughout the process

  • Solution: Check for endogenous proteases that might specifically target the linker region

  • Solution: Design constructs with different linker sequences between protein and tag

• Protein Precipitation During Concentration:

  • Solution: Use gentle concentration methods (dialysis against high-MW PEG instead of centrifugal concentrators)

  • Solution: Keep protein concentration below critical micelle concentration

  • Solution: Include appropriate detergent and glycerol in buffers during concentration

Problem 4: Poor Functional Activity

Symptoms:

• Purified protein shows little or no expected biochemical activity

• Loss of characteristic structural features

Possible Causes and Solutions:

• Detergent-Induced Conformational Changes:

  • Solution: Test alternative, milder detergents or lipid nanodiscs

  • Solution: Include lipids from native source during purification

  • Solution: Attempt reconstitution into liposomes for functional assays

• Loss of Essential Cofactors:

  • Solution: Supplement buffers with potential cofactors (metal ions, lipids)

  • Solution: Analyze native bacterial membranes to identify co-purifying factors

  • Solution: Consider whether protein requires assembly with other ATP synthase components for stability

• Oxidation of Critical Residues:

  • Solution: Include reducing agents (DTT, β-mercaptoethanol) in all buffers

  • Solution: Perform purification under anaerobic conditions if possible

  • Solution: Consider site-directed mutagenesis of problematic cysteine residues if they're not essential for function

Problem 5: Storage Instability

Symptoms:

• Activity loss during storage

• Visible precipitation upon thawing

Possible Causes and Solutions:

• Freeze-Thaw Damage:

  • Solution: Add 50% glycerol to storage buffer as cryoprotectant

  • Solution: Aliquot protein into single-use volumes to avoid repeated freeze-thaw cycles

  • Solution: Store working aliquots at 4°C for up to one week rather than freezing/thawing

• Detergent Precipitation at Low Temperatures:

  • Solution: Use detergents with lower critical micelle temperatures

  • Solution: Increase detergent concentration slightly above CMC in storage buffer

  • Solution: Consider alternative storage methods (lyophilization with appropriate excipients)

• Protein Oxidation During Storage:

  • Solution: Flush storage containers with nitrogen before sealing

  • Solution: Include additional reducing agents in storage buffer

  • Solution: Use amber tubes or wrap in foil if light sensitivity is suspected

These troubleshooting approaches are based on established practices in membrane protein biochemistry and have been successfully applied to similar challenging proteins, including recombinant proteins from Bacillus species.

What are the critical considerations for optimizing PCR conditions when using atpE as a target for bacterial detection?

Optimizing PCR conditions for atpE-based bacterial detection requires careful attention to several critical parameters to ensure reliable, sensitive, and specific results. The following comprehensive guide addresses key considerations based on research findings:

Primer Design Optimization

Critical Considerations:

• Target Region Selection: The atpE gene contains both conserved and variable regions. For genus-specific detection, target conserved regions; for species-specific detection like B. thuringiensis subsp. konkukian, target unique sequence variations .

• Primer Length and Stability: Optimal primers for atpE amplification should be 18-24 nucleotides. Research indicates that primers exceeding 24 bases demonstrate higher detection rates for challenging targets like atpE .

• Terminal Nucleotides: Evidence suggests that primers with G or C at the 3' terminus show stronger binding to the target site, which is particularly important for atpE detection. Primers ending with A or T bases may form more fragile bonds with the target .

Troubleshooting Strategy:

• If experiencing poor amplification, redesign primers to include G/C-rich 3' ends

• For weak signals, consider extending primer length to 22-24 nucleotides

• Test multiple primer pairs targeting different regions of the atpE gene

PCR Reaction Components

Critical Considerations:

Troubleshooting Strategy:

• Perform a magnesium titration (1.0-4.0 mM) to determine optimal concentration

• Test different polymerases if experiencing non-specific amplification

• Consider adding PCR enhancers (DMSO, betaine) at 5-10% for GC-rich regions

Thermal Cycling Parameters

Critical Considerations:

• Initial Denaturation: Thorough initial denaturation is crucial for atpE amplification. Research suggests 95°C for 5 minutes is effective for bacterial samples.

• Annealing Temperature: This is perhaps the most critical parameter for successful atpE amplification:

  • Too low: Increases non-specific amplification

  • Too high: Reduces primer binding and amplification efficiency

  • Recommended approach: Use gradient PCR to determine optimal temperature, typically 2-5°C below calculated primer Tm

• Extension Time: For the typical atpE amplicon size (100-400 bp), 30 seconds at 72°C is usually sufficient.

• Cycle Number: For standard detection, 30-35 cycles is optimal; for low template amounts, up to 40 cycles may be necessary.

Troubleshooting Strategy:

• Perform gradient PCR (±5°C around calculated Tm) to determine optimal annealing temperature

• If non-specific bands appear, increase annealing temperature in 1°C increments

• For weak signals, increase cycle number but be aware of increased risk of non-specific amplification

Template Preparation

Critical Considerations:

• DNA Extraction Method: Research comparing boiling versus chemical extraction methods for atpE detection shows significant differences in sensitivity. Commercial DNA extraction kits generally provide higher quality template than crude lysate methods .

• Template Quantity: For atpE detection, 1-50 ng of bacterial DNA per reaction is typically sufficient. Excessive template can inhibit PCR.

• Template Quality: DNA purity is crucial for successful amplification. Contaminants from extraction can inhibit PCR.

Troubleshooting Strategy:

• If experiencing inconsistent results, compare different DNA extraction methods

• For complex samples (clinical/environmental), include additional purification steps

• Include internal amplification controls to detect inhibition

• Consider diluting template if inhibition is suspected

Real-Time PCR Optimization

Critical Considerations:

• Probe Design: For TaqMan-based detection, probes should target conserved regions within the amplicon with minimal secondary structure.

• Fluorophore Selection: Select dyes and quenchers with minimal spectral overlap if multiplexing.

• Cycling Parameters: For real-time detection of atpE, two-step PCR (combined annealing/extension) at 60°C often provides optimal results.

Troubleshooting Strategy:

• If experiencing poor signal, adjust probe concentration (increase to 100-250 nM)

• Optimize probe position within the amplicon if possible

• Ensure sufficient extension time for complete probe hydrolysis

Validation Approach

Recommended Validation Protocol:

• Analytical Sensitivity: Determine limit of detection using serial dilutions of genomic DNA

• Analytical Specificity: Test with closely related species (e.g., other Bacillus species)

• Reproducibility: Assess intra- and inter-assay variation coefficients

• Robustness: Test with variable template concentrations and quality

Performance Benchmarks:
Published research indicates that optimized atpE-based detection systems can achieve specificity of up to 100% with sensitivity around 61.54% for challenging targets . These metrics provide a benchmark for evaluating your optimization efforts.

By systematically addressing these critical considerations, researchers can develop robust and reliable PCR protocols for atpE-based detection of Bacillus thuringiensis and other target bacteria in diverse sample types.

How does atpE from Bacillus thuringiensis subsp. konkukian compare structurally and functionally with homologs from other bacterial species?

A comprehensive comparison of ATP synthase subunit c (atpE) from Bacillus thuringiensis subsp. konkukian with homologs from other bacterial species reveals important insights into structural conservation, functional differences, and evolutionary relationships:

Primary Sequence Comparison:

The atpE protein from B. thuringiensis subsp. konkukian consists of 72 amino acids (MSLGVIAAAIAIGLSALGAGIGNGLIVSRTIEGVARQPELKGALQTIMFIGVALVEALPIIGVVIAFIVMNK) . When compared with homologs from other bacterial species, several patterns emerge:

Table 1: Primary Sequence Comparison of atpE Across Selected Bacterial Species

Key observations from sequence analysis:

• The atpE from B. thuringiensis subsp. konkukian shows highest similarity to other Bacillus species, particularly B. subtilis (approximately 85-90% sequence identity) .

• The protein is significantly shorter than mycobacterial homologs, primarily due to N-terminal differences .

• Despite length and sequence variations, the core functional regions, especially transmembrane domains and the critical proton-binding site, show high conservation across diverse bacteria .

Structural Features and Functional Implications:

1. Transmembrane Organization:

• B. thuringiensis subsp. konkukian atpE contains two hydrophobic transmembrane α-helices connected by a polar loop, similar to other bacterial homologs .

• The helical regions are highly conserved across species due to their critical role in forming the c-ring structure.

• The connecting loop regions show greater sequence variability between species, reflecting their exposure to the aqueous environment and lesser structural constraints.

2. Oligomeric Assembly:

• The c-subunits form ring-like structures in the membrane with the number of subunits varying across species:

  • Most Bacillus species: 10-11 subunits per c-ring

  • E. coli: 10 subunits

  • Some alkaliphilic bacteria: 13 subunits

• This variability in c-ring stoichiometry affects the bioenergetic properties of ATP synthase, particularly the H⁺/ATP ratio .

3. Proton Binding Site:

• All bacterial atpE proteins contain a conserved acidic residue (typically aspartate or glutamate) that is essential for proton translocation.

• In B. thuringiensis, like other Bacillus species, this critical residue is positioned similarly to the Asp61 in other well-studied species.

• The microenvironment around this residue is highly conserved, ensuring proper pKa and proton-binding capabilities.

4. Species-Specific Adaptations:

• Thermophilic bacteria show specific adaptations in atpE that enhance thermostability.

• Alkaliphilic Bacillus species have modifications that allow function at high pH.

• Mycobacteria possess specific sequence features that enable selective targeting by drugs like diarylquinolines .

Functional Differences and Evolutionary Implications:

1. Bioenergetic Properties:

• The c-ring size directly determines the H⁺/ATP ratio, affecting the bioenergetic efficiency of the organism.

• Species living in energy-limited environments often have larger c-rings (more subunits), allowing ATP synthesis at lower proton motive force.

• B. thuringiensis, as a soil-dwelling organism with variable energetic resources, has a c-ring size optimized for its ecological niche.

2. Drug Susceptibility Patterns:

• The atpE protein is the target of diarylquinoline drugs in mycobacteria.

• Specific residues like Ala63 in M. tuberculosis (when mutated to Pro) confer resistance .

• B. thuringiensis subsp. konkukian atpE lacks some of these critical residues in the equivalent positions, potentially affecting its susceptibility to similar compounds.

• Some species like M. xenopi show natural resistance due to specific amino acid substitutions at otherwise conserved positions .

3. Evolutionary Conservation:

• The high conservation of core functional elements across diverse bacterial phyla indicates strong selective pressure on atpE.

• The variations in non-critical regions reflect adaptation to specific ecological niches and physiological requirements.

• The phylogenetic analysis of atpE sequences generally aligns with established bacterial taxonomy, suggesting it could be used for identification purposes in certain contexts .

This comparative analysis highlights both the fundamental conservation of atpE structure and function across diverse bacterial species and the subtle variations that reflect species-specific adaptations to different ecological niches and physiological requirements.

How do different expression systems and purification methods affect the yield and quality of recombinant atpE protein?

Different expression systems and purification methodologies significantly impact both the yield and functional quality of recombinant ATP synthase subunit c (atpE) from Bacillus thuringiensis subsp. konkukian. This comparative analysis evaluates various approaches based on evidence from similar membrane proteins:

Expression Systems Comparison:

Table 2: Comparison of Expression Systems for Recombinant atpE Production

Key observations:

• Standard E. coli BL21(DE3) systems typically provide the highest raw yield but often at the expense of proper folding and function for membrane proteins like atpE .

• Specialized strains like C41/C43 offer better functional quality by reducing toxicity and improving membrane insertion.

• Alternative systems like cell-free expression provide superior functional quality but at significantly higher cost and reduced scalability.

Expression Conditions Impact:

The specific conditions used within any expression system dramatically affect both yield and quality:

• Temperature Effects:

  • Lower temperatures (16-20°C) generally improve proper folding and membrane insertion

  • Higher temperatures (37°C) maximize expression rate but often lead to inclusion body formation

  • For atpE, expression at 18°C for 16-20 hours typically provides the best balance between yield and quality

• Inducer Concentration:

  • High IPTG concentrations (>0.5 mM) maximize expression but can overwhelm folding machinery

  • Lower concentrations (0.1-0.3 mM) often improve the proportion of properly folded protein

  • For membrane proteins like atpE, lower inducer concentrations combined with longer expression times often yield better results

• Media Composition:

  • Rich media (LB, TB) provide higher biomass but sometimes lower quality protein

  • Defined media allow better control and more consistent results

  • Supplementation with specific lipids can improve membrane protein folding and insertion

Purification Methods Comparison:

Table 3: Comparison of Purification Strategies for Recombinant atpE

Key purification considerations:

Comparative Case Study from Literature:

The expression and purification of ATP synthase components from various Bacillus species illustrates these principles. In the case of ATP-binding proteins from related systems, research has shown:

• Expression in E. coli BL21 with induction by 0.5-1 mM IPTG yielded sufficient protein for biochemical characterization .

• Purification using Ni-NTA affinity chromatography followed by additional labeling steps allowed specific detection and functional analysis .

• Proper buffer composition was critical for maintaining activity during purification and storage .

Recommendations Based on Research Evidence:

• For Structural Studies:

  • Expression: E. coli C41/C43 at 18°C with 0.2 mM IPTG induction

  • Purification: DDM solubilization followed by IMAC and size exclusion chromatography

  • Storage: Tris-based buffer with 50% glycerol at -80°C

• For Functional Studies:

  • Expression: Cell-free system with direct incorporation into nanodiscs

  • Purification: Gentle methods maintaining native-like environment

  • Storage: At 4°C for short term in buffer with minimal detergent

• For High-Throughput Screening:

  • Expression: Standard E. coli BL21(DE3)

  • Purification: Simplified IMAC protocol with single detergent

  • Storage: Small aliquots at -20°C with glycerol to avoid freeze-thaw cycles

These evidence-based recommendations provide a framework for selecting appropriate expression and purification strategies based on the specific requirements of different research applications involving recombinant Bacillus thuringiensis subsp. konkukian atpE protein.