Recombinant Ochrobactrum anthropi Lectin-like protein BA14k (Oant_3884)

What is the Ochrobactrum anthropi Lectin-like protein BA14k (Oant_3884) and what are its key properties?

Ochrobactrum anthropi Lectin-like protein BA14k (Oant_3884) is a 151-amino acid protein with a molecular mass of approximately 17.7 kDa that belongs to the BA14k protein family. The protein possesses immunoglobulin-binding and hemagglutination properties and demonstrates mannose-binding capabilities. It is considered essential for virulence in O. anthropi and may play significant roles in lipopolysaccharide (LPS) biosynthesis or polysaccharide transport mechanisms based on similarity analyses with related proteins . The protein is of particular interest to researchers studying bacterial virulence factors and carbohydrate-binding proteins in pathogenic microorganisms.

What expression systems have been successfully used for recombinant production of Oant_3884?

Recombinant Oant_3884 has been successfully expressed in Escherichia coli expression systems. The mature protein sequence (residues 27-151) is typically used for recombinant expression, often with an N-terminal histidine tag to facilitate purification. E. coli-based expression systems provide several advantages for Oant_3884 production, including high protein yield, cost-effectiveness, and well-established protocols for induction and harvesting . When designing expression constructs, researchers should consider codon optimization for E. coli to enhance expression efficiency, as the GC content and codon usage in Ochrobactrum anthropi differs from E. coli. Additionally, inclusion of appropriate signal sequences may be necessary if the protein requires specific folding environments or post-translational modifications.

What are the optimal purification strategies for recombinant Oant_3884?

Purification of His-tagged recombinant Oant_3884 typically employs immobilized metal affinity chromatography (IMAC) using nickel or cobalt resins. The general protocol involves:

• Cell lysis under native conditions using either sonication or chemical lysis buffers

• Clarification of lysate by centrifugation (typically 10,000-15,000×g for 30 minutes)

• Affinity purification using a nickel or cobalt column with imidazole gradient elution

• Size exclusion chromatography as a polishing step to achieve >90% purity

For applications requiring higher purity, additional chromatographic steps such as ion exchange chromatography may be necessary. The purified protein is typically obtained as a lyophilized powder after buffer exchange and freeze-drying processes . When designing purification protocols, researchers should consider including protease inhibitors to prevent degradation during the purification process, especially since the protein contains several potential protease cleavage sites.

How can the mannose-binding activity of Oant_3884 be assayed in laboratory settings?

The mannose-binding activity of Oant_3884 can be assessed using several complementary approaches:

• Hemagglutination assays: These involve incubating the purified protein with erythrocytes (typically rabbit or human) that present mannose-containing glycans on their surface. Hemagglutination is observed microscopically and quantified by serial dilution to determine the minimum concentration required for activity. Inhibition studies using free mannose can confirm specificity .

• Surface plasmon resonance (SPR): This technique measures real-time binding kinetics between immobilized mannose derivatives and the flowing protein. By analyzing association and dissociation rates, binding constants (KD) can be determined.

• Isothermal titration calorimetry (ITC): This provides thermodynamic parameters of binding (ΔH, ΔS, and KD) by measuring heat changes during interaction between the protein and mannose.

• Fluorescence-based assays: Using fluorescently labeled mannose derivatives to quantify binding through changes in fluorescence intensity or anisotropy upon protein interaction.

When conducting these assays, it's important to include appropriate controls, such as testing binding to other sugars (glucose, galactose) to verify specificity, and using known mannose-binding lectins (e.g., Concanavalin A) as positive controls.

What roles does Oant_3884 play in virulence, and how can these be experimentally verified?

Oant_3884 is reported to be essential for virulence in Ochrobactrum anthropi . Its potential roles in virulence can be experimentally verified through multiple approaches:

• Gene knockout studies: Creating Oant_3884 deletion mutants in O. anthropi and assessing changes in virulence in appropriate infection models. Complementation studies with the wild-type gene should restore virulence phenotypes.

• Host cell adhesion and invasion assays: Comparing wild-type and Oant_3884-deficient strains for their ability to adhere to and invade host cells, particularly those with mannose-rich glycoproteins on their surface.

• LPS biosynthesis analysis: Since Oant_3884 may be involved in LPS biosynthesis, comparative analysis of LPS profiles between wild-type and mutant strains using techniques like SDS-PAGE followed by silver staining or mass spectrometry can reveal functional impacts.

• Immunoglobulin binding assays: Quantifying the protein's ability to bind host immunoglobulins and potentially evade immune responses using ELISA or immunoprecipitation approaches.

• Polysaccharide transport studies: Tracing labeled polysaccharides to determine if Oant_3884 facilitates their transport across membranes.

These experimental approaches should be complemented by clinical correlation studies, comparing expression levels of Oant_3884 in clinical versus environmental isolates of O. anthropi to establish relevance to human infection scenarios .

How can proteomic approaches be utilized to study Oant_3884 expression and interactions in O. anthropi?

Proteomic approaches offer powerful tools for studying Oant_3884 expression patterns and protein-protein interactions:

• Two-dimensional gel electrophoresis (2-DE): This technique separates O. anthropi proteins based on isoelectric point (pI) and molecular weight. For optimal resolution of Oant_3884, researchers should utilize extended pH gradients (e.g., pH 2.3-11.0) as described for O. anthropi proteome mapping . The protein can be identified in gel spots using mass spectrometry after tryptic digestion.

• Quantitative proteomics: Techniques such as iTRAQ (isobaric tags for relative and absolute quantitation) or SILAC (stable isotope labeling with amino acids in cell culture) can measure differential expression of Oant_3884 under various growth conditions or stress factors.

• Interactomics: Protein-protein interactions can be studied using:

  • Pull-down assays with His-tagged recombinant Oant_3884

  • Co-immunoprecipitation with anti-Oant_3884 antibodies

  • Bacterial two-hybrid systems

  • Cross-linking mass spectrometry to capture transient interactions

• Proteomic 'contigs': Similar to the approach described for O. anthropi , overlapping proteomic windows can be constructed to place Oant_3884 in its functional context within protein expression networks. This involves creating contiguous windows of protein expression using multiple gel images with overlapping pH gradient regions (e.g., pH 4-5 and pH 6-7).

When interpreting proteomic data, researchers should be mindful of the technical challenges in resolving higher molecular weight proteins at extreme pI values, as noted in the literature on O. anthropi proteomics .

What structural biology approaches are most promising for elucidating the three-dimensional structure of Oant_3884?

Several structural biology approaches are applicable for determining the three-dimensional structure of Oant_3884:

• X-ray crystallography: This remains the gold standard for high-resolution protein structures. For successful crystallization, researchers should:

  • Produce highly pure, homogeneous protein (>95% purity)

  • Screen diverse crystallization conditions including various precipitants, buffers, pH values, and additives

  • Consider co-crystallization with mannose or other binding partners

  • Explore surface entropy reduction mutations if initial crystallization attempts fail

• NMR spectroscopy: Suitable for proteins <25 kDa, making it appropriate for the 17.7 kDa Oant_3884. This approach requires:

  • 15N and 13C isotopic labeling during recombinant expression

  • Optimization of buffer conditions for spectral quality

  • High protein concentrations (0.5-1 mM) while preventing aggregation

• Cryo-electron microscopy (cryo-EM): Though traditionally used for larger proteins or complexes, recent advances in detectors and processing algorithms make it increasingly viable for smaller proteins, especially if Oant_3884 forms higher-order assemblies.

• Integrative structural biology: Combining multiple low-resolution techniques such as small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and computational modeling to develop structural models.

For any structural studies, researchers should consider the importance of properly folded protein, which may require optimization of expression and purification protocols to maintain native conformation.

What are the optimal storage and handling conditions for maintaining Oant_3884 stability?

To maintain the stability and activity of recombinant Oant_3884, the following storage and handling practices are recommended:

• Long-term storage: Store lyophilized protein at -20°C to -80°C. Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles .

• Buffer composition: For reconstitution, use Tris/PBS-based buffer with 6% trehalose at pH 8.0. Trehalose serves as a cryoprotectant and stabilizing agent .

• Reconstitution protocol:

  • Centrifuge the vial briefly before opening 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% (50% is recommended) for long-term storage at -20°C/-80°C

• Working aliquots: For frequent use, store working aliquots at 4°C for up to one week to minimize freeze-thaw damage .

• Stability considerations: Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and aggregation. When analyzing samples by SDS-PAGE, the addition of reducing agents like DTT or β-mercaptoethanol should be optimized as they may affect the protein's functional properties if disulfide bonds are present.

What are the common pitfalls in recombinant Oant_3884 expression and how can they be addressed?

Several challenges may arise during recombinant expression of Oant_3884, including:

• Inclusion body formation: If the protein forms inclusion bodies in E. coli, strategies to address this include:

  • Lowering expression temperature (16-20°C)

  • Reducing inducer concentration

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Using E. coli strains designed for difficult proteins (e.g., Rosetta, Origami)

  • Adding fusion tags that enhance solubility (e.g., MBP, SUMO)

• Proteolytic degradation: If degradation occurs during expression or purification:

  • Include protease inhibitor cocktails during lysis and purification

  • Consider using E. coli strains deficient in specific proteases (e.g., BL21)

  • Optimize induction time to harvest before degradation becomes significant

• Low expression yield: To improve yields:

  • Optimize codon usage for E. coli

  • Test different promoter systems (T7, tac, etc.)

  • Explore various media formulations, including auto-induction media

  • Optimize cell density at induction and post-induction incubation time

• Protein misfolding: If the protein is expressed but inactive:

  • Consider periplasmic expression strategies

  • Test refolding protocols from solubilized inclusion bodies

  • Explore different buffer compositions during purification

• Endotoxin contamination: For applications requiring endotoxin-free preparations:

  • Use endotoxin removal columns during purification

  • Consider expression in specialized endotoxin-free systems

Monitoring protein expression through time-course sampling and analysis by SDS-PAGE can help identify optimal harvest times and troubleshoot expression issues early in the process.

How does Oant_3884 compare structurally and functionally with other lectins and members of the BA14k family?

Oant_3884 belongs to the BA14k family of proteins and shares several structural and functional features with other lectins, though with distinctive characteristics:

The immunoglobulin-binding property of Oant_3884 is of particular interest as it suggests a potential role in immune evasion that distinguishes it from many classical lectins. This dual functionality (carbohydrate binding and immunoglobulin interaction) presents unique opportunities for structural and functional studies targeting these distinct binding sites.

What insights can be gained from studying Oant_3884 in the context of Ochrobactrum anthropi pathogenesis and identification?

Studying Oant_3884 provides valuable insights into O. anthropi pathogenesis and bacterial identification:

• Species identification: O. anthropi is one of only three species in the Ochrobactrum genus reported in clinical samples, alongside O. intermedium and O. pseudintermedium . The recA gene sequence analysis used for species identification has shown 100% identity with several O. anthropi strains , suggesting Oant_3884 could serve as a potential biomarker for identification.

• Biochemical profiling: Traditional biochemical tests for O. anthropi identification include urease (URE), malate (MAL), and nitrate reduction (NIT) reactions . Understanding how Oant_3884 expression correlates with these biochemical properties could enhance diagnostic accuracy.

• Habitat colonization: O. anthropi colonizes an exceptionally wide variety of habitats . Oant_3884's potential role in adhesion and polysaccharide transport may explain this adaptability, offering insights into bacterial adaptation mechanisms.

• Clinical relevance: As O. anthropi is an emerging opportunistic pathogen, studying virulence factors like Oant_3884 is crucial for understanding clinical manifestations and developing targeted treatments. The lectin's mannose-binding properties may be particularly relevant for interactions with mammalian cell surfaces, which often display mannose-containing glycoproteins.

• Evolutionary perspective: Comparative analysis of Oant_3884 with homologous proteins in related species can reveal evolutionary adaptations and horizontal gene transfer events that have shaped the pathogenic potential of O. anthropi. This may also help explain why O. anthropi has emerged as a clinical isolate while many related species remain environmental.

Research on Oant_3884 thus bridges fundamental bacterial biology with clinical microbiology, offering both basic science insights and potential applications in diagnostics and therapeutics.

What are the potential applications of Oant_3884 in glycobiology research?

Oant_3884's mannose-binding properties position it as a valuable tool in glycobiology research with several promising applications:

• Glycan profiling: Due to its specific carbohydrate-binding properties, purified Oant_3884 could be developed as a probe for detecting and analyzing mannose-containing glycoconjugates in complex biological samples. This application would require:

  • Fluorescent or enzymatic labeling of purified Oant_3884

  • Validation against known mannose-rich glycoproteins

  • Development of standardized binding assays with quantifiable outputs

• Cell surface glycome analysis: The protein could be used to identify and map mannose-containing structures on cell surfaces, potentially revealing differences between normal and pathological states. This approach would complement existing tools like plant lectins in glycobiology research.

• Affinity chromatography: Immobilized Oant_3884 could serve as a selective matrix for purifying mannose-rich glycoproteins from complex mixtures, offering complementary selectivity to existing lectin affinity columns.

• Structural studies of protein-carbohydrate interactions: Co-crystallization of Oant_3884 with defined mannose-containing oligosaccharides could provide atomic-level insights into the structural basis of carbohydrate recognition, potentially revealing novel binding motifs.

• Biosensor development: The protein could be incorporated into biosensing platforms for detecting specific glycan structures in medical diagnostics or quality control applications.

When developing these applications, researchers should carefully characterize the exact binding specificity of Oant_3884, as mannose-binding lectins can show preferences for specific linkages and presentations of mannose residues within complex glycans.

How might structural and functional studies of Oant_3884 contribute to antimicrobial development strategies?

Structural and functional studies of Oant_3884 could inform novel antimicrobial strategies through several approaches:

• Target-based drug design: If Oant_3884 is indeed essential for virulence in O. anthropi , detailed structural information about its active sites could enable rational design of inhibitors that specifically block its function, potentially reducing bacterial virulence without directly killing bacteria (anti-virulence approach).

• Polysaccharide transport disruption: If the protein's role in polysaccharide transport is confirmed, disrupting this function could compromise bacterial cell wall integrity or biofilm formation. This approach would require:

  • Validation of transport function through in vitro assays

  • Identification of critical residues involved in transport

  • Design of small molecules targeting these specific sites

• Lectin-based diagnostics: Knowledge of Oant_3884's binding properties could enable development of rapid diagnostic tests for O. anthropi infections, allowing earlier intervention with appropriate antibiotics.

• Vaccine development: If surface-exposed, Oant_3884 could potentially serve as an antigen for vaccine development, particularly if antibodies against it neutralize its virulence-associated functions.

• Cross-species applications: Insights from Oant_3884 structure-function relationships might apply to homologous proteins in other pathogenic bacteria, broadening the impact of this research.

These approaches align with current efforts to develop alternatives to conventional antibiotics, addressing the growing challenge of antimicrobial resistance. Particularly promising is the anti-virulence strategy, which may exert less selective pressure for resistance development compared to traditional bactericidal approaches.

What proteomics approaches are most suitable for studying post-translational modifications of Oant_3884?

Investigating potential post-translational modifications (PTMs) of Oant_3884 requires sophisticated proteomics approaches:

• Sample preparation strategies:

  • Enrichment techniques specific to anticipated PTMs (e.g., phosphopeptide enrichment using titanium dioxide, glycopeptide enrichment using lectin affinity)

  • Optimized protein extraction from O. anthropi using established proteomic 'contig' approaches with extended pH gradients (2.3-11.0) for maximum coverage

  • Careful preservation of modification status through use of inhibitors (phosphatase inhibitors, deacetylase inhibitors) during extraction

• Mass spectrometry approaches:

  • High-resolution LC-MS/MS using instruments with electron transfer dissociation (ETD) capabilities, which better preserve labile modifications

  • Multiple fragmentation techniques (CID, HCD, ETD) to maximize identification of different PTM types

  • Data-dependent acquisition for discovery, followed by targeted methods (parallel reaction monitoring, PRM) for verification of specific modifications

• Bioinformatic analysis:

  • Search algorithms optimized for PTM discovery (e.g., ModifiComb, PTMfinder)

  • Site localization scoring to confirm modification positions

  • Statistical validation using false discovery rate approaches

  • Integration with structural data to assess functional significance

• Quantitative approaches:

  • SILAC or TMT labeling to quantify PTM dynamics under different conditions

  • Label-free quantification with internal standards for relative abundance measurements

This multi-layered approach would enable comprehensive characterization of the PTM landscape of Oant_3884, including both the types of modifications present and their sites within the protein sequence. Such information is crucial for understanding how PTMs might regulate the protein's activity, localization, or interactions in response to environmental signals.

What are the key considerations for designing knockout and complementation studies to investigate Oant_3884 function in O. anthropi?

Designing effective genetic manipulation studies for Oant_3884 requires careful planning:

• Knockout strategy design:

  • Homologous recombination approach using suicide vectors carrying antibiotic resistance markers flanked by genomic sequences upstream and downstream of Oant_3884

  • CRISPR-Cas9 system adapted for O. anthropi, if available, for precise gene editing

  • Confirmation of knockout by both PCR verification and Western blotting to ensure complete protein elimination

  • Creation of unmarked deletions to minimize polar effects on adjacent genes

• Complementation design:

  • Reintroduction of wild-type Oant_3884 under native promoter control

  • Use of integrative plasmids for stable expression at defined copy number

  • Inclusion of epitope tags for detection while ensuring tag doesn't interfere with function

  • Construction of point mutants targeting predicted functional residues for structure-function studies

• Expression control considerations:

  • Inducible expression systems to modulate Oant_3884 levels

  • Promoter strength evaluation to ensure physiologically relevant expression levels

  • Verification of transcription by RT-qPCR and translation by Western blotting

• Phenotypic analysis pipeline:

  • Growth curve analysis under various conditions

  • Biofilm formation assays

  • Mannose-binding activity using hemagglutination tests

  • Virulence assessment in appropriate cell culture or animal models

  • Polysaccharide transport assays if implicated in LPS biosynthesis

• Controls and validation:

  • Creation of multiple independent knockout clones to rule out secondary mutations

  • Inclusion of controls for potential polar effects on downstream genes

  • Complementation with empty vector as negative control

  • Analysis of global effects using transcriptomics or proteomics to identify compensatory responses

When interpreting results from such studies, researchers should consider potential redundancy in function with other proteins, which might mask phenotypes in single gene knockout studies. Combining knockouts of functionally related genes might be necessary to observe clear phenotypes in some cases.

How might systems biology approaches integrate Oant_3884 into broader understanding of O. anthropi pathogenesis?

Systems biology offers powerful frameworks for contextualizing Oant_3884 within the broader molecular networks of O. anthropi pathogenesis:

• Multi-omics integration:

  • Combination of proteomics 'contigs' approach with transcriptomics, metabolomics, and phenotypic data

  • Network analysis to identify functional modules that include Oant_3884

  • Correlation of Oant_3884 expression patterns with global cellular responses to environmental stimuli

• Protein-protein interaction networks:

  • High-throughput interactomics using affinity purification-mass spectrometry with tagged Oant_3884

  • Validation of key interactions through techniques like bimolecular fluorescence complementation

  • Construction of functional interaction maps centered on Oant_3884

• Regulatory network analysis:

  • Identification of transcription factors controlling Oant_3884 expression

  • Characterization of post-transcriptional regulation mechanisms

  • Integration with stress response pathways and virulence regulation networks

• Host-pathogen interaction modeling:

  • Systems-level analysis of how Oant_3884 contributes to the interface between O. anthropi and host cells

  • Computational prediction of host targets for Oant_3884 binding

  • Integration with host immune response networks

• Comparative systems biology:

  • Cross-species comparison of BA14k family proteins and their regulatory contexts

  • Evolutionary analysis of network motifs involving lectin-like proteins

  • Identification of conserved and divergent features that may explain species-specific pathogenic traits

These approaches would provide a holistic view of how Oant_3884 functions within the complex cellular machinery of O. anthropi, potentially revealing unexpected connections and emergent properties that cannot be discovered through reductionist approaches alone.

What technological advances might enhance our ability to study Oant_3884 structure-function relationships?

Emerging technologies offer exciting opportunities to gain deeper insights into Oant_3884 structure-function relationships:

• Advances in structural biology:

  • Microcrystal electron diffraction (MicroED) for structural determination from nanocrystals

  • Time-resolved crystallography to capture conformational changes during binding events

  • AlphaFold2 and other AI-based structure prediction methods to model protein dynamics

  • Single-particle cryo-EM advancements enabling structural determination of smaller proteins

• High-resolution imaging approaches:

  • Super-resolution microscopy to visualize Oant_3884 localization within bacterial cells

  • Correlative light and electron microscopy (CLEM) to connect functional observations with ultrastructural details

  • Expansion microscopy for improved spatial resolution of protein distribution

• Single-molecule techniques:

  • Atomic force microscopy to measure binding forces between Oant_3884 and its targets

  • Single-molecule FRET to observe conformational changes during binding events

  • Optical tweezers to quantify mechanical properties of Oant_3884-ligand interactions

• Advanced biophysical methods:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map binding interfaces

  • Native mass spectrometry to analyze complexes under near-physiological conditions

  • Micro-scale thermophoresis for quantitative binding studies with minimal material requirements

• Genetic and genome engineering:

  • CRISPR-Cas9 screens to identify genetic interactions with Oant_3884

  • Site-specific incorporation of non-canonical amino acids for precise functional probing

  • Optogenetic control of Oant_3884 expression or activity for temporal studies

The integration of these technologies with traditional biochemical and microbiological approaches would enable unprecedented insights into how the structure of Oant_3884 relates to its diverse functions in mannose binding, immunoglobulin interactions, and potential roles in polysaccharide transport and LPS biosynthesis.