Recombinant Helicobacter pylori Uncharacterized protein HP_0085 (HP_0085)
Description
Introduction to Helicobacter pylori Uncharacterized Protein HP_0085
Helicobacter pylori is a gram-negative bacterium known to colonize the human gastric mucosa, causing various gastroduodenal diseases including gastritis, peptic ulcers, and being a risk factor for gastric cancer. The HP_0085 protein is classified as an uncharacterized protein within the H. pylori proteome, indicating that its precise biological function remains to be elucidated. Hypothetical proteins like HP_0085 constitute a substantial fraction of proteomes in both prokaryotes and eukaryotes, with a majority of them found in bacteria such as H. pylori . The scientific interest in these uncharacterized proteins has increased significantly with advancements in genome sequencing technologies, as they potentially represent novel functional elements within bacterial systems.
The recombinant version of the HP_0085 protein refers to the artificially produced form of this protein, typically expressed in laboratory settings using expression systems such as Escherichia coli. This recombinant form enables researchers to study the protein's properties and functions in controlled environments, facilitating a deeper understanding of its potential role in H. pylori biology and pathogenesis. The protein is identified in databases by its UniProt ID P64651, providing a standardized reference point for researchers investigating this particular protein .
Significance in Helicobacter pylori Research
Studying uncharacterized proteins such as HP_0085 is critical for comprehensive understanding of H. pylori's biology. H. pylori has been extensively studied due to its prevalence in human gastric infections, with numerous strains isolated and sequenced from patients with various clinical manifestations including gastritis, gastric ulcers, and duodenal ulcers . The bacterium demonstrates remarkable genetic diversity, with each isolate typically separated by relatively large unique branches on phylogenetic trees, indicating substantial genetic variation between strains. In this context, understanding the function of every protein, including uncharacterized ones like HP_0085, becomes essential for deciphering strain-specific characteristics and virulence factors.
Hypothetical Proteins: Classification and Challenges
HP_0085 belongs to the broader category of hypothetical proteins, which are predicted proteins for which there is no experimental evidence of translation. Within this category, proteins conserved among organisms from several phylogenetic lineages, but still lacking functional validation, are termed "conserved hypothetical proteins" (CHPs) . The annotation and characterization of such proteins present significant challenges to researchers, requiring multidisciplinary approaches combining computational predictions with experimental validation. Genome sequencing has dramatically increased our awareness of novel genes with unpredictable functions, though putative functions can be predicted for only a fraction of them.
Primary Structure Analysis
Analysis of the primary structure of HP_0085 reveals several notable features. The amino acid composition appears to include a substantial proportion of hydrophobic residues, particularly in the central region of the sequence, suggesting potential membrane association or transmembrane domains. The presence of charged residues at the N and C termini may indicate regions involved in protein-protein interactions or specific functional activities. The relatively short length of the protein (62 amino acids) classifies it as a small protein, potentially functioning as a regulatory peptide, a structural component, or a participant in protein complexes.
Predicted Secondary and Tertiary Structures
While specific experimental data on the secondary and tertiary structures of HP_0085 are not available from the search results, computational prediction methods commonly used for hypothetical proteins might suggest structural elements like alpha helices, beta sheets, or disordered regions . Such predictions can provide initial insights into the protein's potential functional roles. The analysis of uncharacterized proteins generally involves homology-based structure prediction, physiochemical property assessment, and domain analysis, which collectively contribute to functional hypotheses.
Recombinant Protein Design
The recombinant form of HP_0085 has been engineered with an N-terminal His tag to facilitate purification and detection . This modified version represents the full-length protein (amino acids 1-62) with the addition of the His tag sequence at the N-terminus. The His tag serves as an affinity tag for purification using metal affinity chromatography, allowing for efficient isolation of the protein from cellular extracts. This design element is critical for producing high-purity protein samples suitable for biochemical and structural studies.
Biochemical Properties and Expression
The recombinant HP_0085 protein exhibits specific biochemical properties that are important for its handling and application in research settings. Understanding these properties is essential for researchers working with this protein.
Expression and Purification System
The recombinant HP_0085 protein is expressed in Escherichia coli, a common prokaryotic expression system used for producing recombinant proteins . E. coli offers several advantages for protein expression, including rapid growth, high protein yields, and well-established protocols for genetic manipulation. For the purification of recombinant proteins like HP_0085, various techniques can be employed, with the His tag facilitating purification through immobilized metal affinity chromatography (IMAC). The purity of the commercially available recombinant HP_0085 exceeds 90% as determined by SDS-PAGE analysis , indicating a high-quality preparation suitable for research applications.
Physical and Chemical Properties
The recombinant HP_0085 protein is provided as a lyophilized powder, a form that enhances stability during storage . Upon reconstitution, it is recommended to prepare the protein at a concentration of 0.1-1.0 mg/mL in deionized sterile water. For long-term storage, adding glycerol to a final concentration of 5-50% is suggested, with 50% being the default recommendation . These storage conditions aim to preserve the protein's structural integrity and functional properties.
The protein is stored in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . This buffer composition is designed to maintain protein stability by providing appropriate ionic strength and pH conditions. Trehalose, a non-reducing disaccharide, acts as a protein stabilizer, protecting the protein during freeze-drying and storage in the lyophilized state.
Research Status and Applications
The field of hypothetical protein research, including studies of proteins like HP_0085, has evolved significantly with advances in genomic and proteomic technologies. Understanding the potential applications and current research status of HP_0085 provides context for its significance in H. pylori biology.
Functional Prediction Approaches
For uncharacterized proteins like HP_0085, functional prediction typically employs a multi-faceted approach combining computational and experimental methods. Computational methods include homology-based function prediction, whereby similarities to characterized proteins suggest potential functions, and structure-based function prediction, where predicted structural features inform functional hypotheses . Experimental validation often follows, using techniques such as gene expression analysis, protein-protein interaction studies, and phenotypic analysis of knockout or overexpression mutants.
The annotation of hypothetical proteins from pathogenic microorganisms like H. pylori is particularly important because it may reveal new therapeutic targets or provide insights into pathogenesis mechanisms . Proteins like HP_0085 could potentially serve as markers or pharmacological targets for drug design, discovery, and screening, contributing to efforts to combat H. pylori infections and associated diseases.
Experimental Techniques for Characterization
The characterization of uncharacterized proteins typically employs a range of experimental techniques. Mass spectrometry serves as a crucial analytical technique for validating protein characterization, providing information on the protein's mass, sequence, and potential post-translational modifications . Two-dimensional gel electrophoresis (2-DGE) with immobilized pH gradients (IPGs) combined with mass spectrometry represents a core technology for proteomics, especially for studying protein expression, activity, regulation, and modifications at the cellular level .
For recombinant proteins like HP_0085, additional techniques might include circular dichroism spectroscopy for secondary structure determination, nuclear magnetic resonance spectroscopy or X-ray crystallography for tertiary structure determination, and functional assays based on predicted activities. These techniques collectively contribute to a comprehensive understanding of the protein's structural and functional properties.
Reconstitution Protocol
The recommended reconstitution protocol for HP_0085 involves dissolving the lyophilized powder in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . For long-term storage, adding glycerol to a final concentration of 5-50% (with 50% being the default) is suggested. This glycerol addition helps prevent freeze damage and maintain protein solubility during frozen storage.
Care should be taken to avoid repeated freeze-thaw cycles, as these can lead to protein denaturation and loss of activity . Proper reconstitution and storage practices significantly impact the protein's stability and functionality in experimental settings.
Product Specs
Note: While we strive to ship the format currently in stock, we understand that you may have specific requirements. Please indicate your preferred format when placing your order, and we will accommodate your needs to the best of our ability.
Note: Our proteins are standardly shipped with normal blue ice packs. If dry ice shipping is required, please inform us in advance as additional fees may apply.
Generally, the shelf life of the liquid form is 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: heo:C694_00415
STRING: 85962.HP0085
Q&A
What is HP_0085 and what do we currently know about its characteristics?
HP_0085 is an uncharacterized protein from Helicobacter pylori strain ATCC 700392/26695 (formerly known as Campylobacter pylori). Based on available data, this protein:
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Has UniProt accession number P64651
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Consists of 62 amino acids in its full sequence: MQKEQEAQEIAKKAVKIVFFLGLVVVLLMMINLYLINQINASAQMSHQIKKIEERLNQEQK
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Contains hydrophobic regions suggestive of membrane association
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Has not been extensively characterized for function, unlike other H. pylori proteins such as UreG
The amino acid sequence analysis suggests HP_0085 may be a membrane-associated protein, which has implications for expression strategies and purification approaches.
What expression systems are most effective for producing recombinant H. pylori proteins?
The selection of expression systems for H. pylori proteins depends on the specific protein characteristics:
Escherichia coli systems:
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The most commonly used system for initial expression attempts due to its simplicity and cost-effectiveness
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BL21(DE3) and its derivatives are frequently used for cytoplasmic expression
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For potentially toxic membrane proteins like HP_0085, tightly regulated systems such as BL21(DE3)pLysS or Lemo21(DE3) may be preferable
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Fusion tags such as His6, GST, or MBP can improve solubility and facilitate purification
Alternative expression systems:
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Yeast systems (P. pastoris, S. cerevisiae) for proteins requiring eukaryotic post-translational modifications
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Insect cell expression (baculovirus) for complex membrane proteins
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Cell-free expression systems for toxic proteins
For HP_0085 specifically, E. coli expression with optimization for membrane proteins would be a reasonable starting point, similar to approaches used for other H. pylori proteins .
What purification strategies are recommended for recombinant HP_0085?
For membrane-associated proteins like HP_0085, purification typically involves:
Extraction strategies:
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Mild detergents (DDM, LDAO, or Triton X-100) for membrane protein solubilization
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Sequential extraction methods to separate peripheral from integral membrane proteins
Purification methods:
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Affinity chromatography (typically Ni-NTA for His-tagged proteins) as seen with other H. pylori recombinant proteins
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Size exclusion chromatography for further purification and buffer exchange
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Ion exchange chromatography as an additional purification step
Specific considerations for HP_0085:
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Based on protocols for other H. pylori proteins, purification yields of 1-3 mg/L of culture can be expected
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Storage in Tris-based buffer with 50% glycerol as indicated for the commercially available recombinant protein
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Avoiding repeated freeze-thaw cycles to maintain protein integrity
What are the optimal conditions for enhancing soluble expression of membrane-associated H. pylori proteins?
For potentially membrane-associated proteins like HP_0085, several strategies can enhance soluble expression:
Temperature optimization:
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Lower expression temperatures (15-25°C) often improve folding and solubility
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Extended expression times (overnight to 24 hours) at lower temperatures
Media and induction parameters:
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Rich media (TB or 2xYT) supplemented with glucose for improved biomass
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Low IPTG concentrations (0.1-0.5 mM) for slower, more controlled expression
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Auto-induction media for gradual protein expression without monitoring
Solubility enhancement strategies:
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Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ) to assist proper folding
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Fusion with solubility-enhancing tags (SUMO, MBP, TrxA)
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Screening multiple constructs with varying N- and C-terminal boundaries
For H. pylori proteins specifically, the UreG expression protocol demonstrated successful expression in both soluble and insoluble forms, with higher yields in the insoluble fraction (3 mg/L vs 1.4 mg/L for soluble protein) .
How can experimental design approaches improve recombinant protein expression outcomes?
Systematic experimental design can significantly enhance expression optimization:
Design of Experiments (DoE) approach:
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Factorial design to simultaneously evaluate multiple parameters (temperature, inducer concentration, media composition)
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Response surface methodology to identify optimal conditions based on yield and solubility
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Statistical analysis to determine significant factors affecting expression
Implementation strategy:
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Define critical quality attributes (yield, purity, solubility, activity)
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Identify factors potentially affecting these attributes
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Design screening experiments covering multiple factors
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Perform optimization experiments focusing on significant factors
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Validate optimized conditions
This systematic approach, as referenced in the literature for other recombinant proteins, can reduce development time and improve protein quality compared to one-factor-at-a-time optimization .
What experimental approaches can be used to elucidate the function of uncharacterized H. pylori proteins?
For uncharacterized proteins like HP_0085, a multi-faceted approach is recommended:
Bioinformatic analysis:
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Sequence homology and conserved domain searches
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Structural prediction using tools like AlphaFold
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Genomic context and co-expression analysis
Biochemical characterization:
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Protein-protein interaction studies (pull-down assays, two-hybrid systems)
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Enzymatic activity screens based on predicted function
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Lipid binding assays (if membrane association is predicted)
Immunological approaches:
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Production of polyclonal antibodies against the recombinant protein
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Testing reactivity with sera from H. pylori-infected patients
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Western blot analysis of native expression in different H. pylori strains
This comprehensive approach has been successful for other H. pylori proteins such as UreG, which showed good immunological reactivity and diagnostic potential .
How does HP_0085 compare to other H. pylori proteins as a potential diagnostic marker?
While specific data for HP_0085 is limited, comparison can be made based on approaches used for other H. pylori proteins:
Evaluation criteria for diagnostic potential:
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Prevalence across H. pylori strains
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Antigenicity in infected patients
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Specificity (absence of cross-reactivity with other pathogens)
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Sensitivity in different disease states
Comparative assessment:
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Well-characterized H. pylori proteins like UreB, VacA, CagA, HspB, FlaA, and FlaB have established diagnostic utility
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UreG specifically showed 70% reactivity with IgG antibodies and 60% with IgA antibodies from infected patients
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Combined IgG/IgA detection improved sensitivity to 83.3% with high specificity (97.5% for IgG, 92.5% for IgA)
For HP_0085 to be evaluated as a diagnostic marker, similar seroreactivity studies would need to be conducted, comparing results to established markers.
What methods can determine the seroreactivity of recombinant HP_0085 with patient samples?
Seroreactivity testing follows established protocols:
Western blot analysis:
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Purified recombinant protein separated by SDS-PAGE and transferred to membranes
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Probing with patient sera followed by HRP-conjugated anti-human IgG and IgA detection
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Comparison between culture-positive H. pylori patients and control groups
ELISA-based detection:
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Direct coating of recombinant protein on microplates
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Titration of patient sera to determine optimal dilution
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Quantitative assessment of antibody levels compared to established cutoff values
Controls and validation:
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Inclusion of multiple control groups: healthy individuals, H. pylori-negative gastric symptom patients, and patients with other diseases
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Production of hyperimmune sera in animal models to validate antigen recognition
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Testing of native protein recognition using bacterial lysates
The approach used for UreG protein, testing 30 positive and 40 control sera with both IgG and IgA detection, provides a methodological framework applicable to HP_0085 .
How can recombinant HP_0085 be incorporated into multiplexed diagnostic platforms?
For improved diagnostic performance, HP_0085 could be incorporated into multiplex platforms:
Protein microarray systems:
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Multiple recombinant H. pylori proteins immobilized on a single platform
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Simultaneous detection of antibodies against various targets
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Machine learning algorithms to interpret complex reactivity patterns
Bead-based multiplex assays:
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Conjugation of different recombinant proteins to uniquely coded microspheres
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Flow cytometry-based detection of multiple antibody specificities
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Higher sensitivity compared to traditional ELISA
Implementation considerations:
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Optimization of coupling chemistry for HP_0085 immobilization
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Determination of optimal protein concentration to avoid saturation
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Cross-reactivity assessment between different protein antigens
This approach aligns with current diagnostic trends seeking to improve sensitivity by combining multiple H. pylori antigens rather than relying on single markers .
What structural characterization methods are appropriate for membrane-associated proteins like HP_0085?
For structural characterization of HP_0085:
Solution-based techniques:
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Circular dichroism (CD) spectroscopy for secondary structure assessment
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Differential scanning calorimetry (DSC) for thermal stability analysis
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Limited proteolysis to identify structured domains
Advanced structural methods:
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X-ray crystallography (challenging for membrane proteins; may require lipidic cubic phase)
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Cryo-electron microscopy for larger complexes
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NMR spectroscopy for smaller membrane proteins or soluble domains
Membrane interaction studies:
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Surface plasmon resonance with immobilized lipid bilayers
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Tryptophan fluorescence for monitoring membrane insertion
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Liposome binding assays to characterize lipid specificity
Given the small size of HP_0085 (62 amino acids) and potential membrane association, NMR spectroscopy of the protein in membrane-mimetic environments might be particularly suitable.
What strategies can identify potential protein-protein interactions of HP_0085?
Identifying interaction partners provides functional insights:
Affinity-based methods:
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Pull-down assays using tagged recombinant HP_0085 as bait
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Co-immunoprecipitation from H. pylori lysates using anti-HP_0085 antibodies
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Chemical cross-linking followed by mass spectrometry (XL-MS)
Library screening approaches:
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Yeast two-hybrid screening against H. pylori genomic libraries
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Phage display to identify peptide binding partners
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Protein complementation assays in bacterial systems
Validation techniques:
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Surface plasmon resonance to determine binding kinetics
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Microscale thermophoresis for affinity quantification
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Co-expression and co-purification of interaction complexes
These methods have successfully identified protein-protein interactions for other H. pylori proteins and could reveal functional associations for HP_0085.
What strategies address the challenges of expressing transmembrane H. pylori proteins in heterologous systems?
Membrane protein expression presents unique challenges:
Expression optimization strategies:
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Use of specialized E. coli strains (C41/C43, derived from BL21) designed for membrane protein expression
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Codon optimization for the expression host
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Fusion with reporter proteins (GFP) to monitor folding and insertion
Detergent screening matrix:
| Detergent Class | Examples | Advantages | Challenges |
|---|---|---|---|
| Mild Non-ionic | DDM, DM, OG | Gentle extraction | Limited efficiency |
| Zwitterionic | LDAO, FC-12 | Efficient extraction | Potential denaturation |
| Peptide-based | SMA copolymers | Native lipid retention | Limited compatibility |
| Amphipols | A8-35 | Stability enhancement | Secondary purification |
Application to HP_0085:
Based on its 62-amino acid sequence with hydrophobic regions, HP_0085 might require specialized membrane protein expression systems or solubilization approaches to maintain native conformation .
How can CRISPR-Cas9 genome editing facilitate functional studies of HP_0085 in H. pylori?
CRISPR-based approaches enable precise functional analysis:
Gene knockout strategies:
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CRISPR-Cas9 delivery via conjugation or natural transformation
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Design of guide RNAs specific to the HP_0085 gene
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Phenotypic characterization of knockout strains under various conditions
Genetic complementation:
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Re-introduction of wild-type or mutant HP_0085 variants
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Controlled expression using native or inducible promoters
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Assessment of functional restoration
Tagged protein expression:
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CRISPR-mediated insertion of epitope tags or fluorescent proteins
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Visualization of protein localization in living bacteria
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Affinity purification of native protein complexes
These approaches could reveal the physiological role of HP_0085 in H. pylori colonization, survival, or pathogenesis, similar to studies conducted for other H. pylori proteins.
How does the immunogenicity of HP_0085 compare to established H. pylori vaccine candidates?
Evaluation of HP_0085 as a vaccine candidate requires systematic comparison:
Immunogenicity assessment:
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Animal immunization studies comparing antibody titers against HP_0085 versus established antigens
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Characterization of antibody subclasses and T-cell responses
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Evaluation of cross-reactivity against different H. pylori strains
Protective efficacy metrics:
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Challenge studies in animal models measuring bacterial load reduction
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Histopathological assessment of gastric inflammation
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Comparison with established protective antigens like UreB
Benchmark comparison:
Several H. pylori proteins have been investigated as vaccine candidates, including UreB, VacA, CagA, HpaA, NapA, FlaA, and FlaB . For HP_0085 to be considered, similar systematic immunological studies would be required, focusing on both humoral and cellular immune responses.
What approaches can determine the role of HP_0085 in H. pylori pathogenesis?
Understanding pathogenic relevance requires multiple approaches:
Colonization studies:
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Comparison of wild-type and HP_0085 knockout strains in colonization models
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Competition assays between wild-type and mutant strains
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Evaluation of bacterial fitness under stress conditions
Host interaction analyses:
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Effect on epithelial cell adhesion and invasion
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Influence on inflammatory cytokine production
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Impact on gastric epithelial cell viability and proliferation
Clinical correlation studies:
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Expression levels of HP_0085 in clinical isolates from different disease states
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Association between HP_0085 variants and disease severity
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Antibody responses against HP_0085 in patients with different H. pylori-associated pathologies
These approaches would determine whether HP_0085 contributes to the various gastric and extragastric manifestations associated with H. pylori infection .
