Recombinant Burkholderia pseudomallei Protease HtpX homolog (htpX)
Description
Overview of Recombinant Burkholderia pseudomallei Protease HtpX Homolog (htpX)
The HtpX protease homolog is a zinc-dependent metalloprotease involved in protein quality control under stress conditions. The recombinant version is engineered to include an N-terminal His tag for purification and detection purposes. It is expressed in Escherichia coli and corresponds to the full-length protein (1–285 amino acids) encoded by the htpX gene (UniProt ID: Q3JXD9) from B. pseudomallei strain 1710b .
Production and Purification
The protein is produced via recombinant DNA technology, with the following workflow:
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Cloning: The htpX gene is inserted into an expression vector under a strong promoter.
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Expression: Induced in E. coli under optimized conditions.
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Purification: Affinity chromatography using Ni-NTA resin to exploit the His tag .
Yield: Typical yields range from 0.5–2 mg/L of culture, depending on expression conditions .
Applications in Research
Recombinant HtpX is primarily used in:
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Immunoassays: As an antigen in ELISA to detect B. pseudomallei-specific antibodies .
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Functional Studies: Investigating its role in bacterial stress adaptation and virulence regulation.
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Drug Discovery: Screening for inhibitors targeting metalloproteases in pathogenic Burkholderia spp. .
Research Gaps and Future Directions
While recombinant HtpX is well-characterized structurally, its precise biological role in B. pseudomallei pathogenesis remains underexplored. Key unanswered questions include:
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Its interaction with host proteins during infection.
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Regulatory mechanisms under hypoxia or oxidative stress.
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Contribution to antibiotic resistance or biofilm formation.
Further studies leveraging this recombinant protein could clarify its potential as a therapeutic target or diagnostic marker for melioidosis.
Product Specs
Note: While we prioritize shipping the format currently in stock, we can accommodate specific format requirements. Please indicate your preference in the order notes, and we will fulfill your request.
Note: Our proteins are shipped with standard blue ice packs. Should you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, liquid form has a shelf life of 6 months at -20°C/-80°C, while lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: bpl:BURPS1106A_0163
Q&A
How is the htpX gene organized and conserved across different B. pseudomallei strains?
The htpX gene is present in multiple B. pseudomallei strains with high sequence conservation. Based on genomic data, the gene has been mapped to different locus tags in various strains:
Comparative genomic analysis shows remarkable conservation of the htpX gene across B. pseudomallei strains (>99% sequence identity) and significant homology with other Burkholderia species. For example, the HtpX protease from B. phytofirmans shares substantial sequence similarity, indicating evolutionary conservation across the genus . This high conservation suggests the protein likely performs essential functions in bacterial physiology.
What expression systems have proven most effective for recombinant production of B. pseudomallei HtpX?
The most effective expression system documented for recombinant B. pseudomallei HtpX protease is Escherichia coli, with N-terminal His-tag fusions commonly employed to facilitate purification. Expression typically uses:
| Expression Parameter | Recommended Condition |
|---|---|
| Expression host | E. coli BL21(DE3) or similar strains |
| Vector system | pET or pQE series with inducible promoters |
| Fusion tag | N-terminal His6 tag |
| Induction conditions | 0.1-1.0 mM IPTG, 16-25°C for 4-18 hours |
| Media | LB or TB supplemented with appropriate antibiotics |
When expressing membrane proteins like HtpX, researchers should consider using specialized E. coli strains optimized for membrane protein expression (e.g., C41/C43) and lower induction temperatures (16-20°C) to minimize toxicity and inclusion body formation .
What are the optimal purification strategies for obtaining functional B. pseudomallei HtpX protease?
The purification of HtpX presents challenges due to its multiple transmembrane domains. A successful purification strategy includes:
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Cell lysis using detergent-based buffers (e.g., n-dodecyl β-D-maltoside or CHAPS)
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Immobilized metal affinity chromatography (IMAC) utilizing the His-tag
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Size exclusion chromatography for final purification
The recommended storage buffer contains Tris-based buffer with 50% glycerol at pH 8.0, which helps maintain protein stability . Purified protein should be stored at -20°C/-80°C, with working aliquots maintained at 4°C for up to one week. Repeated freeze-thaw cycles significantly reduce enzyme activity and should be avoided .
How does HtpX protease contribute to B. pseudomallei pathogenesis and virulence?
While direct evidence for HtpX's role in B. pseudomallei pathogenesis is limited in current literature, several inferences can be made based on protease functions in this pathogen:
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B. pseudomallei utilizes various proteases as virulence factors to establish infection and evade host immune responses .
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As a metalloprotease, HtpX likely contributes to protein quality control during stress conditions encountered within host cells.
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The bacterium's ability to survive in oxygen-limited environments (like those inside host cells) depends on specific proteases and regulatory systems .
The significance of proteases in B. pseudomallei virulence is underscored by studies showing that specific proteases like MprA can elicit protective immune responses when used as vaccine components . This suggests proteases, potentially including HtpX, are expressed during infection and recognized by the host immune system.
What is the relationship between HtpX and bacterial stress response mechanisms?
HtpX proteases typically function in bacterial stress response pathways. In B. pseudomallei, this protease likely participates in:
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Hypoxic adaptation: B. pseudomallei requires specific regulatory networks to switch from oxygen-dependent respiration to alternative metabolic pathways when invading host tissues. The RegAB two-component system serves as a master regulator in this process . HtpX may assist in remodeling membrane protein composition during this metabolic shift.
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Intracellular survival: As an intracellular pathogen, B. pseudomallei must adapt to the environment within host cells. Proteases can degrade misfolded proteins that accumulate during stress conditions, maintaining bacterial viability .
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Nutrient acquisition: Some bacterial proteases facilitate nutrient acquisition by breaking down host proteins. Whether HtpX contributes to this function remains to be determined.
How might the structure of B. pseudomallei HtpX inform inhibitor design for therapeutic applications?
Analysis of the amino acid sequence reveals that B. pseudomallei HtpX contains conserved metalloprotease motifs, including potential zinc-binding regions necessary for catalytic activity. This structural information could guide inhibitor design through:
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Structure-based drug design: Molecular modeling of the catalytic domain could identify potential binding pockets for small molecule inhibitors.
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Comparative structural analysis: Leveraging similarities and differences between bacterial and human metalloproteases to develop selective inhibitors.
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Peptidomimetic approaches: Designing pseudopeptide inhibitors based on substrate recognition sequences.
Given the increase in multidrug-resistant B. pseudomallei strains, targeting virulence factors like proteases presents an alternative therapeutic strategy that may be less susceptible to conventional resistance mechanisms .
What methodologies are most appropriate for investigating HtpX substrate specificity?
Investigating substrate specificity of B. pseudomallei HtpX requires a multi-faceted approach:
| Methodology | Application to HtpX | Technical Considerations |
|---|---|---|
| Fluorescence-based protease assays | Quantitative measurement of enzyme kinetics | Requires design of specific fluorogenic substrates |
| Proteomic identification of cleavage sites (PICS) | Global identification of cleavage sites | Requires mass spectrometry capabilities |
| Bacterial two-hybrid systems | Protein-protein interactions | Useful for membrane protein interactions |
| Peptidomics analysis | Identification of natural peptide substrates | Combines LC-MS/MS with bioinformatics |
| Site-directed mutagenesis | Validation of catalytic residues | Requires prediction of active site residues |
When designing experiments, researchers should account for the membrane-bound nature of HtpX, which may require detergent-based buffer systems for maintaining enzyme activity in vitro .
How does B. pseudomallei HtpX compare with similar proteases in other bacterial pathogens?
Comparative analysis of B. pseudomallei HtpX with homologs in other bacterial species reveals important insights:
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Conservation across Burkholderia species: The HtpX amino acid sequence shows approximately 99% identity between B. pseudomallei and B. thailandensis , and significant similarity with B. phytofirmans HtpX .
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Functional domains: The HtpX protease maintains conserved metalloprotease domains across bacterial species, with six N-terminal transmembrane domains characteristic of membrane-bound proteases.
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Evolutionary significance: The high conservation of HtpX across diverse bacteria suggests it performs fundamental physiological functions that have been maintained through evolutionary selection.
Understanding these relationships helps position B. pseudomallei HtpX within the broader context of bacterial protease evolution and may provide insights into its fundamental biological roles.
What experimental approaches are recommended for evaluating potential immunogenicity of B. pseudomallei HtpX?
Given that some B. pseudomallei proteases have demonstrated immunogenic properties and vaccine potential , evaluating HtpX immunogenicity would involve:
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In silico epitope prediction: Computational identification of potential B-cell and T-cell epitopes within the HtpX sequence.
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Animal immunization studies: Following the approach used with MprA protease, which generated protective immunity in mouse models .
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Human serum reactivity: Testing recognition of recombinant HtpX by sera from recovered melioidosis patients.
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Cytokine profiling: Measuring immune cell activation and cytokine production in response to purified HtpX protein.
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Vaccination challenge studies: Evaluating protective efficacy using established mouse models of melioidosis.
The approach used with MprA protease, which elicited significant IgG responses predominantly of the IgG1 isotype (indicating a Th2 immune response) and protected mice against lethal challenge , provides a methodological template for similar studies with HtpX.
How might gene knockout studies of htpX inform our understanding of B. pseudomallei virulence mechanisms?
Targeted gene deletion studies of htpX would significantly advance our understanding of this protease's role in B. pseudomallei pathogenesis. Such studies should:
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Generate clean deletion mutants using allelic exchange methods
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Compare the growth of wild-type and ΔhtpX strains under various stress conditions
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Evaluate intracellular survival in macrophage infection models
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Assess virulence in established mouse models of melioidosis
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Examine effects on biofilm formation and antibiotic resistance
Similar approaches with other regulatory systems like RegAB have revealed critical roles in anaerobic adaptation and virulence , suggesting that htpX knockout studies might uncover previously unknown functions in B. pseudomallei biology.
What challenges exist in integrating HtpX into broader proteome-level studies of B. pseudomallei pathogenesis?
Integration of HtpX into comprehensive proteome studies faces several challenges:
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Membrane protein analysis: The membrane-bound nature of HtpX makes it technically challenging to extract and analyze using standard proteomic approaches.
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Conditional expression: HtpX expression may be tightly regulated and only induced under specific environmental conditions, requiring careful experimental design.
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Post-translational modifications: Potential modifications affecting HtpX function may be missed in standard proteomic workflows.
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Redundancy in protease function: B. pseudomallei encodes multiple proteases (80 putative peptidases identified, including 48 serine peptidases and 32 metallopeptidases) , making it difficult to isolate the specific contributions of HtpX.
Future studies should employ targeted approaches combined with global proteomics to overcome these limitations and accurately position HtpX within the complex landscape of B. pseudomallei virulence factors.
