Recombinant Ochrobactrum anthropi ATP synthase subunit a 2 (atpB2)

How is recombinant Ochrobactrum anthropi atpB2 typically expressed and purified for research applications?

Recombinant Ochrobactrum anthropi atpB2 is typically expressed in E. coli expression systems . The process generally follows these methodological steps:

• Gene cloning: The atpB2 gene (Oant_2514) is amplified from O. anthropi genomic DNA and cloned into an appropriate expression vector with an N-terminal His-tag.

• Expression conditions: The recombinant protein is expressed in E. coli using optimized conditions, often employing a tightly regulated expression system to control protein production .

• Purification process:

  • Initial cell lysis under conditions that maintain protein stability

  • Affinity chromatography using the His-tag for primary purification

  • Potential secondary purification steps such as ion-exchange chromatography

  • Final purification by size exclusion chromatography may be employed

• Quality control: The purified protein is assessed for:

  • Purity (>90% as determined by SDS-PAGE)

  • Proper folding and activity

  • Absence of contaminants

• Storage: The purified protein is typically lyophilized or stored in a buffer containing stabilizers such as trehalose (6%) at pH 8.0, and stored at -20°C/-80°C for long-term stability .

What are the optimal storage and handling conditions for recombinant atpB2 protein to maintain stability and activity?

Based on established protocols for recombinant atpB2 protein, the following storage and handling conditions are recommended to maintain optimal stability and activity :

Storage conditions:

• Store lyophilized protein at -20°C/-80°C upon receipt

• For extended storage periods, maintain at -80°C

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

• Avoid repeated freeze-thaw cycles which can significantly reduce protein activity and stability

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is standard) to prevent freeze-thaw damage

• Aliquot into small volumes to minimize freeze-thaw cycles

Buffer conditions:

• Storage buffer typically consists of Tris/PBS-based buffer with 6% trehalose at pH 8.0

• For recombinant protein applications, Tris-based buffer with 50% glycerol is also used

Handling precautions:

• Minimize exposure to room temperature

• Avoid contamination

• Use sterile techniques when preparing aliquots

• Track the number of freeze-thaw cycles for each aliquot

How can Ochrobactrum anthropi atpB2 be used in structural biology studies, and what crystallization methods have proven successful?

While the search results don't provide specific information about crystallization of atpB2, based on general approaches for membrane proteins and ATP synthase components, the following methodological framework would be applicable:

Structural biology approaches for atpB2:

• X-ray crystallography preparation:

  • Detergent screening is critical for membrane protein crystallization

  • Vapor diffusion methods (hanging or sitting drop)

  • Lipidic cubic phase crystallization may be suitable for this membrane protein

  • Screening of various precipitants, pH conditions, and additives

• Cryo-EM analysis:

  • Sample preparation with appropriate detergents or nanodiscs

  • Negative staining for initial assessment

  • Vitrification conditions optimization

  • High-resolution data collection strategies

• NMR studies:

  • Isotopic labeling (15N, 13C) of the recombinant protein

  • Detergent micelle selection for optimal spectral quality

  • 2D and 3D heteronuclear experiments for structural elucidation

• Protein engineering considerations:

  • Creation of truncated constructs removing flexible regions

  • Introduction of mutations to improve crystal contacts

  • Fusion protein approaches (e.g., with T4 lysozyme) to enhance crystallizability

For membrane proteins like atpB2, successful crystallization often requires extensive optimization of detergent conditions and careful consideration of the protein's amphipathic nature. Collaborations with structural biology groups experienced in membrane protein crystallization would be beneficial.

What are the experimental considerations for studying the interaction between atpB2 and other subunits of the ATP synthase complex?

Investigating the interactions between atpB2 and other ATP synthase subunits requires comprehensive experimental approaches:

• Co-immunoprecipitation studies:

  • Use of anti-His antibodies to pull down His-tagged atpB2

  • Western blot analysis to identify co-precipitated subunits

  • Crosslinking prior to co-IP to capture transient interactions

• Bacterial two-hybrid systems:

  • Adaptation of the system for membrane protein interactions

  • Systematic testing of interactions with other ATP synthase subunits

  • Controls to verify specificity of interactions

• Surface plasmon resonance (SPR):

  • Immobilization of His-tagged atpB2 on Ni-NTA sensor chips

  • Measurement of binding kinetics with other purified subunits

  • Determination of binding constants under various conditions

• Förster resonance energy transfer (FRET):

  • Labeling of atpB2 and potential interaction partners

  • Measurement of energy transfer efficiency as indicator of proximity

  • Live-cell FRET to observe interactions in native environment

• Reconstitution experiments:

  • Purification of individual ATP synthase components

  • Systematic reconstitution to identify minimal functional units

  • Activity assays to correlate subunit interactions with function

• Structural analysis:

  • Cross-linking coupled with mass spectrometry to map interaction surfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify binding interfaces

  • Molecular modeling based on known ATP synthase structures

These approaches would provide complementary data on the interaction network of atpB2 within the ATP synthase complex of O. anthropi.

How does the function of atpB2 in Ochrobactrum anthropi compare with ATP synthase subunits in other bacterial species?

While specific comparative data for atpB2 across species is not directly provided in the search results, a methodological framework for such comparison includes:

• Sequence analysis and evolutionary conservation:

  • Multiple sequence alignment of atpB2 with homologous proteins

  • Identification of conserved domains and residues across bacterial species

  • Phylogenetic analysis to determine evolutionary relationships

• Structural comparison:

  • Homology modeling based on known ATP synthase structures

  • Comparison of predicted secondary and tertiary structures

  • Analysis of species-specific structural features

• Functional comparisons:

  • Heterologous expression studies in ATP synthase-deficient strains

  • Complementation assays to test functional interchangeability

  • Measurement of ATP synthesis/hydrolysis rates across species variants

• Adaptation to environmental conditions:

  • Analysis of sequence variations in relation to bacterial habitat

  • Investigation of how adaptations affect ATP synthase function

  • Correlation with O. anthropi's versatile lifestyle and pathogenic potential

• Regulatory mechanisms:

  • Comparison of gene regulation across bacterial species

  • Analysis of operon structures and transcriptional control

  • Investigation of post-translational modifications

These comparative approaches would provide insights into how atpB2 function in O. anthropi relates to ATP synthase function in other bacteria, potentially revealing adaptations specific to O. anthropi's ecological niche and pathogenic potential.

What are the most effective expression conditions for maximizing the yield of soluble recombinant atpB2 protein?

Based on the available information and general principles for membrane protein expression:

• Expression system optimization:

  • E. coli is commonly used for atpB2 expression

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

  • Evaluation of alternative expression hosts (e.g., Pseudomonas, Bacillus) if E. coli yields are insufficient

• Vector and promoter selection:

  • Implementation of tightly regulated expression systems to control protein production

  • Use of re-engineered coliphage T5 promoters containing symmetrical DNA segments that bind efficiently to lactose repressor

  • Inclusion of the lacI(q) gene for precise regulation of expression levels

• Induction parameters:

  • Optimization of IPTG concentration (the degree of induction is controllable by varying inducer concentration)

  • Lower temperatures (16-25°C) during induction to slow protein production and improve folding

  • Extended induction times at lower temperatures

• Media and growth conditions:

  • Evaluation of different media formulations (LB, TB, auto-induction media)

  • Testing of different growth phases for induction (mid-log vs. late-log)

  • Supplementation with specific ions or cofactors that might stabilize the protein

• Solubilization strategies:

  • Screening of detergents for optimal solubilization

  • Co-expression with chaperones to improve folding

  • Use of fusion partners that enhance solubility

Systematic optimization of these parameters through factorial experimental design would help identify conditions that maximize the yield of functional recombinant atpB2.

What troubleshooting approaches are recommended when facing challenges in atpB2 protein expression or purification?

When encountering difficulties with atpB2 expression or purification, consider the following systematic troubleshooting approaches:

Expression Troubleshooting:

• Low expression levels:

  • Verify plasmid sequence integrity

  • Test multiple expression strains

  • Optimize codon usage for E. coli

  • Evaluate alternative promoters or induction systems

  • Consider tightly regulated expression systems as described for O. anthropi

• Protein aggregation/inclusion bodies:

  • Reduce expression temperature (16-20°C)

  • Decrease inducer concentration

  • Co-express with molecular chaperones

  • Test expression as fusion with solubility-enhancing tags

  • Implement slow induction methods (e.g., auto-induction media)

• Protein degradation:

  • Add protease inhibitors during cell lysis

  • Use protease-deficient host strains

  • Modify construct design to remove protease-sensitive regions

  • Optimize lysis and purification buffers

Purification Troubleshooting:

• Poor binding to affinity resin:

  • Verify tag accessibility (consider tag position change)

  • Optimize binding conditions (pH, salt concentration)

  • Test different affinity resins

  • Ensure His-tag is not cleaved during expression

• Impurities in purified protein:

  • Implement additional purification steps (ion exchange, size exclusion)

  • Increase washing stringency

  • Test different elution conditions

  • Consider on-column refolding approaches

• Protein instability after purification:

  • Identify and add stabilizing agents (glycerol, trehalose)

  • Optimize buffer composition and pH

  • Store at appropriate temperature (-20°C/-80°C)

  • Lyophilize protein for long-term storage

• Loss of activity:

  • Verify proper folding using biophysical methods

  • Test activity immediately after purification

  • Optimize storage conditions to maintain activity

  • Consider rapid freezing methods to preserve structure

Systematic documentation of each troubleshooting iteration is essential for identifying patterns and successful approaches.

What are the validated methods for assessing the functionality and activity of purified recombinant atpB2?

While the search results don't provide specific activity assays for atpB2, the following methodological approaches would be applicable based on the protein's function:

• ATP synthesis/hydrolysis assays:

  • Reconstitution of purified atpB2 with other ATP synthase subunits

  • Measurement of ATP synthesis in proteoliposomes under proton gradient

  • Coupled enzyme assays to monitor ATP hydrolysis activity

  • Real-time monitoring using luminescence-based ATP detection

• Proton translocation assays:

  • Reconstitution into liposomes with pH-sensitive fluorescent dyes

  • Measurement of proton flux using pH electrode systems

  • Stopped-flow spectroscopy to measure rapid proton translocation kinetics

  • Patch-clamp electrophysiology for direct measurement of proton currents

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure

  • Fluorescence spectroscopy to assess tertiary structure

  • Differential scanning calorimetry to determine thermal stability

  • Limited proteolysis to probe folding quality

• Binding assays:

  • Surface plasmon resonance to measure interactions with other subunits

  • Isothermal titration calorimetry to determine binding thermodynamics

  • Microscale thermophoresis for quantitative binding analysis

  • Pull-down assays to verify interactions with physiological partners

• Functional complementation:

  • Expression in ATP synthase-deficient bacterial strains

  • Assessment of respiratory growth rescue

  • Measurement of membrane potential restoration

  • Evaluation of ATP synthesis in complemented strains

These methodological approaches provide a comprehensive assessment of atpB2 functionality from multiple perspectives, ensuring that the purified protein retains its native biological activity.

How does the ATP synthase system in Ochrobactrum anthropi compare with other bacterial species, particularly in terms of subunit composition and regulation?

Based on the available information and general knowledge of bacterial ATP synthases:

• Subunit composition comparison:

  • O. anthropi's ATP synthase appears to have two variants of subunit a (atpB1 and atpB2) , which is not uncommon in bacteria but may reflect specific adaptations

  • Comparative genomic analysis would reveal the complete subunit composition

  • The evolutionary relationship between the different a subunits could provide insights into functional specialization

• Gene organization and regulation:

  • O. anthropi possesses tightly regulated expression systems that have been engineered using the lacI(q) gene and re-engineered coliphage T5 promoters

  • This regulatory system allows for controlled expression with up to 57-fold increase when induced

  • The degree of induction can be precisely controlled by varying IPTG concentration

• Evolutionary adaptations:

  • O. anthropi is versatile with strains living in diverse habitats and is increasingly recognized as an opportunistic pathogen

  • Multilocus sequence typing suggests an epidemic population structure with a major clonal complex corresponding to a human-associated lineage

  • These adaptive features may be reflected in its energy metabolism systems, including ATP synthase

• Functional specialization:

  • The presence of multiple atpB variants suggests possible functional specialization

  • Different subunits might be expressed under different environmental conditions

  • Research on purification and characterization of various enzymes from O. anthropi (e.g., purine nucleosidase) demonstrates the organism's metabolic versatility

A systematic comparative genomics and proteomics approach would provide deeper insights into how O. anthropi's ATP synthase system has adapted to its versatile lifestyle and potential pathogenic role.

What biotechnological applications have been developed using recombinant atpB2 or related ATP synthase components?

The search results don't specifically mention biotechnological applications of atpB2, but based on general applications of ATP synthase components and O. anthropi's characteristics, the following applications could be relevant:

• Bioenergy applications:

  • Development of biomimetic energy conversion systems inspired by ATP synthase

  • Engineering of hybrid systems for improved ATP production efficiency

  • Creation of nanomotors based on the rotary mechanism of ATP synthase

• Biosensing platforms:

  • Utilization of ATP synthase components as biological recognition elements

  • Development of proton gradient sensors for environmental monitoring

  • Creation of drug screening platforms targeting energy metabolism

• Therapeutic targets:

  • Design of inhibitors targeting bacterial ATP synthase for antimicrobial development

  • Research into O. anthropi as an opportunistic pathogen in hospitalized patients suggests ATP synthase components could be drug targets

  • Identification of species-specific features for selective targeting

• Protein engineering:

  • Structure-guided engineering of ATP synthase components for enhanced stability

  • Development of chimeric proteins with novel functions

  • Design of minimal ATP synthase systems for synthetic biology applications

• Bioremediation and environmental applications:

  • Exploitation of O. anthropi's versatility for bioremediation processes

  • Development of engineered strains with modified energy metabolism for specific applications

  • Creation of biosensors for environmental monitoring based on ATP synthase activity

Research in these areas would benefit from the availability of well-characterized recombinant proteins like atpB2 and the development of expression systems for O. anthropi as described in the search results .

What approaches can be used to study the role of atpB2 in Ochrobactrum anthropi pathogenicity and its potential as a therapeutic target?

Given that O. anthropi is increasingly recognized as an opportunistic pathogen in hospitalized patients , investigating atpB2's role in pathogenicity involves:

• Gene knockout and complementation studies:

  • Creation of atpB2 deletion mutants using targeted gene replacement

  • Phenotypic characterization of mutants under various growth conditions

  • Complementation studies to confirm gene-phenotype relationships

  • Use of the tightly regulated expression system developed for O. anthropi to control gene expression levels

• Virulence assessment:

  • Infection models to compare wild-type and atpB2 mutant strains

  • Measurement of bacterial survival in host-mimicking conditions

  • Assessment of biofilm formation and antibiotic resistance

  • Evaluation of host immune response to wild-type vs. mutant strains

• Structural analysis for drug targeting:

  • Identification of atpB2 structural features distinct from human ATP synthase

  • In silico screening for compounds binding to unique bacterial epitopes

  • Structure-activity relationship studies of potential inhibitors

  • Crystallization and structural determination to guide drug design

• Population biology approaches:

  • Analysis of atpB2 sequence variation among clinical isolates

  • Correlation of sequence variants with pathogenicity or host adaptation

  • Application of multilocus sequence typing to identify lineages with enhanced virulence

  • Genomic fingerprinting by pulsed-field gel electrophoresis to distinguish isolates

• Host-pathogen interaction studies:

  • Investigation of host immune recognition of atpB2

  • Analysis of atpB2 expression during different infection stages

  • Identification of potential post-translational modifications affecting virulence

  • Study of atpB2's potential role in adaptation to the host environment

These approaches would provide comprehensive insights into atpB2's role in O. anthropi pathogenicity and evaluate its potential as a therapeutic target for this emerging opportunistic pathogen.

What is the recommended protocol for site-directed mutagenesis of atpB2 to study structure-function relationships?

The following detailed protocol outlines the recommended approach for site-directed mutagenesis of atpB2:

Materials Required:

• Recombinant plasmid containing atpB2 gene

• High-fidelity DNA polymerase (e.g., Pfu Ultra, Q5)

• Mutagenic primers (forward and reverse)

• DpnI restriction enzyme

• Competent E. coli cells

• Selection antibiotics

• PCR thermal cycler

• DNA purification kits

Protocol Steps:

• Primer Design:

  • Design complementary primers containing the desired mutation

  • Ensure 15-20 bases of perfect complementarity on each side of the mutation

  • Verify primer properties (Tm, GC content, secondary structures)

  • Consider codon optimization for E. coli expression

• DpnI Digestion:

  • Add 1 μl DpnI (10 units) directly to PCR product

  • Incubate at 37°C for 1-2 hours to digest methylated parental DNA

  • Heat-inactivate DpnI at 80°C for 20 minutes

• Transformation:

  • Transform 5 μl of DpnI-treated product into competent E. coli cells

  • Plate on selective media containing appropriate antibiotics

  • Incubate at 37°C overnight

• Colony Screening:

  • Pick 5-10 colonies for plasmid isolation

  • Perform restriction analysis to verify plasmid integrity

  • Sequence the entire atpB2 gene to confirm:

    • Presence of the desired mutation

    • Absence of unwanted mutations

• Protein Expression Verification:

  • Express mutant protein using the validated expression system for O. anthropi proteins

  • Verify expression by SDS-PAGE and Western blot

  • Compare expression levels with wild-type protein

• Functional Analysis:

  • Purify mutant protein using established protocols

  • Conduct activity assays to determine effect of mutation

  • Perform structural analysis to detect conformational changes

This comprehensive protocol ensures reliable generation and verification of atpB2 mutants for structure-function studies.

What are the most effective methods for studying the membrane integration and topology of atpB2?

To study the membrane integration and topology of atpB2, the following methodological approaches are recommended:

• Computational prediction analysis:

  • Hydropathy plot analysis to identify potential transmembrane segments

  • Topology prediction algorithms (TMHMM, TOPCONS, Phobius)

  • Signal peptide prediction (SignalP)

  • Comparison with known ATP synthase subunit a structures

• Biochemical approaches:

  • Protease protection assays:

    • Express atpB2 in membrane vesicles

    • Treat with proteases in presence/absence of detergents

    • Analyze protected fragments by mass spectrometry

    • Identify regions exposed to cytoplasm vs. periplasm

  • Chemical labeling:

    • Use membrane-impermeable biotinylation reagents

    • Identify accessible lysine or cysteine residues

    • Compare labeling patterns in intact cells vs. permeabilized cells

    • Map labeled residues to structural models

• Genetic approaches:

  • Reporter fusion analysis:

    • Create series of C-terminal truncations fused to reporter proteins

    • Use dual reporters (e.g., GFP, PhoA) with opposite topology requirements

    • Systematically map membrane-spanning segments

    • Validate with site-directed mutagenesis of key residues

  • Substituted cysteine accessibility method (SCAM):

    • Replace native cysteines with alanines

    • Introduce single cysteines at positions of interest

    • Probe with membrane-permeable/impermeable sulfhydryl reagents

    • Determine accessibility from each side of membrane

• Biophysical methods:

  • FRET analysis:

    • Label specific residues with fluorescent pairs

    • Measure energy transfer efficiency

    • Determine distances between labeled positions

    • Map to structural models

  • EPR spectroscopy:

    • Introduce spin labels at specific positions

    • Measure accessibility to paramagnetic reagents

    • Determine depth of residues in membrane

    • Develop constraint-based structural models

• Direct structural analysis:

  • Cryo-EM of reconstituted complexes:

    • Reconstitute purified atpB2 into nanodiscs or liposomes

    • Determine structure by cryo-EM

    • Map transmembrane helices and orientations

    • Compare with existing ATP synthase structures

These complementary approaches provide comprehensive insights into atpB2 membrane topology and integration, essential for understanding its structure-function relationship.

What are the recommended procedures for reconstituting atpB2 into liposomes for functional studies?

The following detailed protocol outlines the recommended procedure for reconstituting atpB2 into liposomes for functional studies:

Materials Required:

• Purified recombinant atpB2 protein

• Synthetic phospholipids (e.g., POPC, POPE, E. coli total lipid extract)

• Detergents (e.g., n-dodecyl-β-D-maltoside, Triton X-100)

• Bio-Beads SM-2 or other detergent adsorbents

• Buffer components (HEPES, KCl, MgCl₂)

• Ultracentrifuge and rotors

• Extrusion apparatus with polycarbonate filters

• Fluorescent probes for functional assays

Reconstitution Protocol:

This detailed protocol provides a methodological foundation for reconstituting atpB2 into liposomes for functional studies, with considerations for protein stability based on the handling recommendations for the recombinant protein .