Recombinant Haemophilus influenzae UPF0761 membrane protein CGSHiGG_04180 (CGSHiGG_04180)

What is UPF0761 membrane protein CGSHiGG_04180 and what are its basic properties?

UPF0761 membrane protein CGSHiGG_04180 is a 269-amino acid membrane protein isolated from Haemophilus influenzae strain PittGG. The protein has a UniProt ID of A5UG94 and is part of the UPF0761 protein family. The full amino acid sequence is: MISLKNFGLLFWKRFSENKLNQVAGALTYSTMLAMVPLVMVIFSIFSAFPVFNEVTGELKEMIFTNFAPSASDMVGEYIDQFVSNSKKMSAVGIVSLIAVALMLINNIDRTLNSIWHNSQSRSPLSSFAIYWMILTLGPLIIGVSIGISSYIKIMFEQSEHLSLGLKLLSFVPFLFTWFIFTLIYTVVPNKKVKIKHSAYGAFLAAIFFTLGKQAFTWYIVTFPSYQLIYGAMATLPIML LWIQISWLVVLVGAQLASTLDEIGEQIEQ . As a membrane protein, it contains hydrophobic regions that facilitate its integration into the bacterial membrane, with multiple transmembrane domains predicted from its sequence.

The protein's molecular weight is approximately 16.6 kDa, similar to other outer membrane proteins of H. influenzae that have been studied. Its function has not been fully characterized, though structural analysis suggests it plays a role in membrane integrity or transport functions typical of bacterial membrane proteins.

How does UPF0761 membrane protein differ from other H. influenzae membrane proteins?

Unlike the well-characterized P6 outer membrane protein of H. influenzae (which has been extensively studied as a vaccine candidate), UPF0761 membrane protein CGSHiGG_04180 belongs to a different protein family with distinct structural features . While P6 is a 16,600-dalton lipoprotein present in both typeable and nontypeable strains of H. influenzae with a cleaved signal sequence resulting in a mature protein of 134 amino acids, UPF0761 is a 269-amino acid protein with different transmembrane topology .

The sequence analysis shows that UPF0761 lacks the characteristic lipoprotein signal sequence found in P6, suggesting a different membrane anchoring mechanism . Additionally, UPF0761 lacks the extensive homology with bacterial lipoproteins that P6 demonstrates, indicating potentially different functions within the bacterial membrane architecture. While P6 has been established as an important antigen in immunity to H. influenzae, the immunogenic properties of UPF0761 remain to be fully characterized.

What expression systems are available for producing recombinant UPF0761 membrane protein?

Multiple expression systems have been developed for the production of recombinant UPF0761 membrane protein. The most commonly used system is E. coli, which provides high protein yields and is relatively simple to work with . When expressing in E. coli, the full-length protein (amino acids 1-269) is typically fused to an N-terminal His-tag to facilitate purification .

Alternative expression systems include yeast, baculovirus, and mammalian cell expression systems . Each system offers distinct advantages depending on research needs:

The choice of expression system should be determined by the specific experimental requirements, including the need for post-translational modifications, protein folding constraints, and downstream applications.

What are the optimal purification protocols for recombinant UPF0761 membrane protein?

Purification of recombinant UPF0761 membrane protein typically employs affinity chromatography utilizing the His-tag fusion. A recommended methodological approach includes:

• Cell Lysis: Bacterial cells expressing the recombinant protein should be lysed using either sonication or French press in a buffer containing mild detergents to solubilize the membrane protein (typically Tris-based buffer with 0.1-1% detergent such as n-dodecyl-β-D-maltoside).

• Immobilized Metal Affinity Chromatography (IMAC): The lysate is applied to a nickel or cobalt affinity column that binds the His-tagged protein. After washing with increasing imidazole concentrations (20-50 mM) to remove non-specifically bound proteins, the target protein is eluted with high imidazole (250-500 mM) .

• Size Exclusion Chromatography: For higher purity (>95%), a second purification step using size exclusion chromatography is recommended to remove aggregates and contaminating proteins.

• Quality Assessment: Purity should be assessed by SDS-PAGE, with typical preparations achieving >90% purity . Western blot analysis using antibodies against the His-tag or the protein itself can confirm identity.

• Detergent Exchange: If necessary for downstream applications, the purified protein can be exchanged into different detergents through dialysis or during size exclusion chromatography.

The critical aspect of this purification strategy is maintaining the membrane protein in a properly folded state throughout the process, which requires careful selection of detergents and buffer conditions.

How should researchers optimize storage conditions for UPF0761 membrane protein stability?

To maintain stability of recombinant UPF0761 membrane protein, specific storage conditions are essential. Based on experimental data, the following methodological approach is recommended:

• Buffer Composition: Store the purified protein in a Tris-based buffer (typically pH 8.0) containing 50% glycerol to prevent freeze-damage to protein structure . The buffer may be supplemented with 6% trehalose as a cryoprotectant .

• Temperature Considerations: For long-term storage, maintain the protein at -20°C or preferably -80°C . For working stocks, aliquots can be stored at 4°C for up to one week .

• Aliquoting Strategy: It is critically important to aliquot the protein solution into single-use volumes before freezing to avoid repeated freeze-thaw cycles, which significantly decrease protein stability and activity .

• Reconstitution Protocol: For lyophilized protein preparations, briefly centrifuge the vial 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% (typical recommendation is 50%) and aliquot for long-term storage .

• Quality Control: Before each use, verify protein integrity by SDS-PAGE or functional assays to ensure the storage conditions have preserved the protein's structure and activity.

These methodological approaches have been empirically determined to maintain >90% protein integrity over storage periods of several months when properly implemented.

What analytical techniques are most informative for structural characterization of UPF0761 membrane protein?

For comprehensive structural characterization of UPF0761 membrane protein, researchers should employ a multi-technique approach:

• Circular Dichroism (CD) Spectroscopy: This technique provides valuable information about secondary structure content (α-helices, β-sheets) and can confirm proper folding of the recombinant protein. For membrane proteins like UPF0761, modified CD protocols using detergent-solubilized protein samples are recommended.

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): This approach determines the oligomeric state of the protein in solution and can detect aggregation or complex formation. For membrane proteins, the technique must account for the detergent micelle contribution to the molecular weight.

• Limited Proteolysis coupled with Mass Spectrometry: This technique identifies flexible regions and domain boundaries within the protein structure. Comparing proteolytic patterns between native and recombinant protein can validate structural integrity.

• Cryo-Electron Microscopy: For high-resolution structural analysis, cryo-EM has become a preferred method for membrane proteins that are difficult to crystallize. Sample preparation typically involves reconstitution into nanodiscs or vitrification in detergent micelles.

• X-ray Crystallography: Though challenging for membrane proteins, this remains the gold standard for atomic-resolution structures. Specialized crystallization techniques using lipidic cubic phases or bicelles may be necessary.

Each of these methods provides complementary information, and their combined use can yield a comprehensive structural understanding of UPF0761 membrane protein. The methodological challenges specific to membrane proteins must be addressed through careful optimization of detergent conditions and sample preparation protocols.

How can UPF0761 membrane protein be evaluated for potential vaccine development?

Evaluation of UPF0761 membrane protein as a vaccine candidate requires a systematic research approach:

• Antigenicity Assessment: First, researchers should determine the antigenicity of purified recombinant UPF0761 protein using sera from patients recovered from H. influenzae infections. ELISA assays can quantify antibody binding, while epitope mapping can identify immunodominant regions. This approach mirrors successful studies with other H. influenzae membrane proteins like P6 .

• Conservation Analysis: Genomic comparison across H. influenzae strains is essential to determine sequence conservation of UPF0761. A highly conserved protein presents a better vaccine target. Bioinformatic analysis should examine sequence variation among clinical isolates from different geographical regions, similar to studies conducted on outer membrane protein subtypes .

• Animal Immunization Studies: A methodological approach for animal studies should include:

  • Immunization with purified recombinant protein using appropriate adjuvants

  • Determination of antibody titers and specificity

  • Challenge studies with virulent H. influenzae strains

  • Evaluation of protection levels and immune correlates

• Structural Vaccinology: Advanced structure-based vaccine design may involve:

  • Identification of surface-exposed epitopes through structural analysis

  • Design of chimeric proteins or peptide vaccines incorporating multiple epitopes

  • Optimization of antigen presentation through particle-based delivery systems

• Cross-Protection Assessment: Determine if antibodies raised against UPF0761 provide protection against different H. influenzae serotypes, particularly focusing on typeable versus non-typeable strains .

The experimental approach should be comparable to established methods used for other H. influenzae outer membrane proteins that have progressed to vaccine development.

What approaches are most effective for studying protein-protein interactions involving UPF0761 membrane protein?

Investigating protein-protein interactions of UPF0761 membrane protein requires specialized techniques suitable for membrane proteins:

• Bacterial Two-Hybrid Systems Modified for Membrane Proteins: Traditional yeast two-hybrid systems are often ineffective for membrane proteins. Instead, bacterial-based systems like BACTH (Bacterial Adenylate Cyclase Two-Hybrid) that allow membrane protein expression should be employed to screen for potential interaction partners.

• Co-Immunoprecipitation with Membrane-Specific Protocols: This approach requires:

  • Crosslinking of protein complexes in vivo prior to cell lysis

  • Solubilization using mild detergents that preserve protein-protein interactions

  • Immunoprecipitation using antibodies against UPF0761 or its fusion tag

  • Mass spectrometry analysis of co-precipitated proteins

• Surface Plasmon Resonance (SPR) Analysis: For kinetic analysis of specific interactions:

  • Immobilize purified UPF0761 on a sensor chip in a lipid environment

  • Flow potential interacting proteins and measure binding kinetics

  • Determine association and dissociation constants for identified interactions

• Microscale Thermophoresis (MST): This technique allows detection of interactions in solution with minimal protein consumption:

  • Fluorescently label UPF0761 protein

  • Mix with potential binding partners at various concentrations

  • Measure changes in thermophoretic mobility to determine binding affinities

• Biolayer Interferometry with Liposome Reconstitution: This method allows study of membrane protein interactions in a lipid environment:

  • Reconstitute UPF0761 into liposomes

  • Immobilize liposomes on sensors

  • Measure interference patterns as potential binding partners interact

Each method provides different information about protein interactions, and complementary approaches should be used to validate findings. Special consideration must be given to maintaining the native structure of UPF0761 membrane protein throughout these experiments.

How can researchers investigate the functional role of UPF0761 membrane protein in H. influenzae?

To elucidate the functional role of UPF0761 membrane protein in H. influenzae, researchers should implement a comprehensive functional genomics approach:

• Gene Knockout/Knockdown Studies: Create isogenic mutants of H. influenzae lacking the gene encoding UPF0761:

  • Use homologous recombination or CRISPR-Cas9 technology for gene deletion

  • Compare growth rates, morphology, and stress responses of wild-type and mutant strains

  • Perform complementation studies to confirm phenotype specificity

• Transcriptomic and Proteomic Analysis: Examine global changes in gene expression and protein abundance:

  • Compare RNA-seq profiles between wild-type and UPF0761 mutants

  • Perform quantitative proteomics to identify compensatory changes in protein expression

  • Analyze under various stress conditions (temperature, pH, antibiotic pressure)

• Membrane Permeability and Transport Studies:

  • Measure membrane integrity using fluorescent dyes and permeability assays

  • Assess transport of specific substances across the membrane

  • Evaluate changes in membrane potential in the presence/absence of UPF0761

• Host-Pathogen Interaction Models:

  • Compare adhesion and invasion of wild-type and mutant strains in cell culture models

  • Assess immune response elicitation by wild-type vs. mutant strains

  • Evaluate virulence in appropriate animal models of H. influenzae infection

• Structural-Functional Correlations:

  • Create site-directed mutants altering key residues identified through structural analysis

  • Assess the impact of these mutations on protein function and bacterial physiology

  • Use labeled protein to track localization during different growth phases and stress conditions

This multifaceted approach provides complementary lines of evidence to establish the physiological role of UPF0761 membrane protein in H. influenzae biology and pathogenesis.

What controls should be included when working with recombinant UPF0761 membrane protein?

Robust experimental design for studies involving recombinant UPF0761 membrane protein requires comprehensive controls:

• Expression System Controls:

  • Empty vector control: Host cells transformed with expression vector lacking the UPF0761 gene

  • Positive expression control: Expression vector containing a well-characterized membrane protein

  • Growth condition controls: Parallel cultures with and without induction to confirm specific expression

• Protein Purity and Integrity Controls:

  • SDS-PAGE analysis: Visualize protein purity (should be >90%)

  • Western blot analysis: Confirm protein identity using anti-His antibodies or specific antibodies against UPF0761

  • Mass spectrometry: Verify protein mass and sequence coverage

  • Size exclusion chromatography: Assess protein homogeneity and oligomeric state

• Functional Assay Controls:

  • Denatured protein control: Heat-denatured UPF0761 to serve as negative control

  • Known membrane protein with similar topology: Comparative control for membrane insertion assays

  • Background activity measurements: Account for non-specific interactions in binding studies

• Stability Controls:

  • Time-course stability assessment: Monitor protein degradation over experimental timeframe

  • Buffer comparison controls: Test multiple buffer conditions to optimize stability

  • Freeze-thaw controls: Assess protein integrity after storage procedures

• Species-Specific Controls:

  • Comparison with native protein: When possible, parallel experiments with native UPF0761 isolated from H. influenzae

  • Cross-species homologs: Comparison with homologous proteins from related bacterial species

Implementation of these controls ensures experimental rigor and facilitates troubleshooting of unexpected results. Particularly important is the verification of proper folding and membrane topology, as recombinant membrane proteins may not always adopt their native conformation.

How can researchers validate the function of recombinant UPF0761 membrane protein?

Validating the function of recombinant UPF0761 membrane protein requires multiple complementary approaches:

• Structure-Based Activity Predictions:

  • Bioinformatic analysis to predict functional domains and active sites

  • Comparison with structurally similar proteins of known function

  • Molecular dynamics simulations to predict functional movements or binding pockets

• Reconstitution into Membrane Models:

  • Proteoliposome reconstitution: Incorporate purified protein into artificial liposomes

  • Planar lipid bilayer experiments: Measure electrical properties and transport activity

  • Nanodiscs: Reconstitute into soluble membrane mimetics for solution-based assays

• Binding and Interaction Studies:

  • Ligand binding assays using purified protein

  • Bacterial surface display assays to evaluate binding to host factors

  • Pull-down assays to identify interaction partners in H. influenzae lysates

• Complementation Studies:

  • Express recombinant UPF0761 in UPF0761-knockout H. influenzae strains

  • Assess restoration of phenotype to validate functional activity

  • Use clinically relevant phenotypic assays (biofilm formation, antibiotic resistance, etc.)

• Comparative Function Analysis with Native Protein:

  • Side-by-side functional assays with native membrane preparations

  • Activity comparison across different expression systems

  • Effect of posttranslational modifications on functional activity

The specific functional assays will depend on the predicted role of UPF0761 based on sequence homology and preliminary data. If UPF0761 functions as a transporter, flux assays would be appropriate; if it plays a structural role, membrane integrity assays would be more relevant.

What are the challenges in crystallizing membrane proteins like UPF0761 and how can they be addressed?

Crystallization of membrane proteins like UPF0761 presents significant challenges that require specialized methodological approaches:

• Protein Stability and Homogeneity Challenges:

  • Challenge: Membrane proteins often aggregate or denature during purification

  • Solution: Screen multiple detergents (n-dodecyl-β-D-maltoside, digitonin, LMNG) to identify optimal stabilizing conditions

  • Methodological approach: Implement thermal stability assays (TSA) to quantitatively compare protein stability in different detergent/lipid combinations

• Crystal Packing Challenges:

  • Challenge: Limited hydrophilic surface area for crystal contacts

  • Solution: Fusion protein approaches with crystallization chaperones (T4 lysozyme, BRIL)

  • Methodological approach: Design construct with fusion partner inserted in a loop region predicted not to impact protein folding

• Detergent Micelle Interference:

  • Challenge: Detergent micelles may prevent crystal contacts

  • Solution: Lipidic cubic phase (LCP) crystallization method

  • Methodological approach: Reconstitute protein in monoolein or related lipids, set up in-meso crystallization trials

• Conformational Heterogeneity:

  • Challenge: Membrane proteins often exist in multiple conformational states

  • Solution: Ligands or antibody fragments to stabilize single conformation

  • Methodological approach: Screen conformation-specific nanobodies or ligands that lock the protein in a defined state

• Alternative Structural Techniques When Crystallization Fails:

  • Challenge: Some membrane proteins resist crystallization despite optimization

  • Solution: Single-particle cryo-electron microscopy (cryo-EM)

  • Methodological approach: Prepare UPF0761 in amphipol or nanodiscs for cryo-EM analysis, particularly if the protein forms oligomers (which increases particle size for imaging)

These methodological approaches have been successfully applied to membrane proteins similar to UPF0761. Implementation requires systematic optimization of each variable and willingness to explore multiple techniques in parallel.

How should researchers approach conflicting results in UPF0761 membrane protein studies?

When confronted with conflicting results in UPF0761 membrane protein studies, researchers should implement a systematic troubleshooting approach:

• Methodological Variation Analysis:

  • Compare experimental protocols in detail, noting differences in:

    • Expression systems and constructs (full-length vs. truncated protein)

    • Purification methods and detergent choices

    • Buffer compositions and pH conditions

    • Protein concentration and oligomeric state

  • Systematically test whether these variations explain the discrepancies

• Protein Quality Assessment:

  • Evaluate protein quality metrics across studies:

    • Purity levels (>90% purity is standard for reliable results)

    • Confirmation of correct folding via spectroscopic methods

    • Batch-to-batch variation documentation

  • Re-purify protein using standardized protocols to eliminate quality issues

• Technical Bias Identification:

  • Examine technical biases that might explain discrepancies:

    • Detection method sensitivity and dynamic range

    • Equipment calibration differences

    • Reagent lot variation

  • Implement blinded analysis protocols to reduce experimenter bias

• Replication Strategy:

  • Design validation experiments that specifically address conflicts:

    • Independent replication by different laboratory members

    • Use of alternative techniques to measure the same parameter

    • Expanded sample sizes to increase statistical power

  • Collaborate with groups reporting conflicting results when possible

• Integrated Data Analysis:

  • Use meta-analysis approaches to integrate conflicting data:

    • Weight results based on methodological robustness

    • Identify consistent trends across variable conditions

    • Develop unified models that explain apparent contradictions

  • Consider whether UPF0761 exists in multiple functional states that could explain divergent results

This methodological framework promotes scientific rigor while acknowledging that conflicting results are often valuable indicators of complex biological phenomena rather than simply experimental errors.

What bioinformatic approaches are most useful for analyzing UPF0761 membrane protein?

Comprehensive bioinformatic analysis of UPF0761 membrane protein requires multiple computational approaches:

• Sequence-Based Structural Prediction:

  • Transmembrane topology prediction using consensus methods (TMHMM, Phobius, TOPCONS)

  • Secondary structure prediction to identify α-helical and β-sheet regions

  • Signal peptide analysis to predict processing and membrane insertion

  • Disorder prediction to identify flexible regions

• Evolutionary Analysis:

  • Multiple sequence alignment of UPF0761 homologs across bacterial species

  • Phylogenetic analysis to understand evolutionary relationships

  • Conservation mapping to identify functionally important residues

  • Coevolution analysis to predict interacting residue pairs

• Structural Bioinformatics:

  • Homology modeling using related proteins with known structures

  • Molecular dynamics simulations in membrane environment

  • Docking simulations to predict potential binding partners

  • Electrostatic surface mapping to identify potential functional sites

• Functional Prediction Tools:

  • Gene neighborhood analysis to identify functionally related genes

  • Gene ontology term enrichment for predicted functions

  • Protein-protein interaction network prediction

  • Integration with transcriptomic data from H. influenzae studies

• Comparative Genomics Approaches:

  • Analysis across H. influenzae strains to identify variants

  • Comparison with membrane proteins of known function in related species

  • Identification of genomic islands or horizontal gene transfer events

These computational methods provide a strong theoretical foundation for experimental design and interpretation. Results from these analyses can be integrated into a comprehensive model of UPF0761 structure and function that guides laboratory experiments.

How can researchers determine whether in vitro findings with recombinant UPF0761 are physiologically relevant?

Establishing physiological relevance of in vitro findings with recombinant UPF0761 membrane protein requires a multi-faceted validation approach:

• Native Protein Comparison Studies:

  • Side-by-side functional assays comparing recombinant protein with native UPF0761 isolated from H. influenzae membranes

  • Validation of protein-protein interactions identified in vitro using co-immunoprecipitation from bacterial lysates

  • Comparison of biochemical properties (stability, oligomerization) between recombinant and native forms

• Genetic Manipulation in H. influenzae:

  • Generation of UPF0761 knockout strains to assess phenotypic changes

  • Complementation studies with wild-type and mutant versions of the protein

  • Site-directed mutagenesis of key residues identified in vitro to confirm their importance in vivo

• Physiologically Relevant Assay Conditions:

  • Adjustment of buffer conditions to mimic the bacterial periplasmic environment

  • Incorporation of relevant ions and metabolites at physiological concentrations

  • Temperature, pH, and osmolarity conditions matching those of H. influenzae natural habitat

• Cell-Based Validation Systems:

  • Expression of UPF0761 variants in heterologous bacterial systems

  • Development of reporter systems linked to UPF0761 function

  • Bacterial two-hybrid or split-protein complementation assays in cellular context

• Correlative Studies with Clinical Isolates:

  • Analysis of UPF0761 expression levels in clinical isolates with varying virulence

  • Correlation of UPF0761 sequence variants with specific phenotypes

  • Functional studies in strains isolated from different infection sites

This methodological framework builds a bridge between controlled in vitro experiments and the complex physiological environment of the bacterial cell. Consistent findings across multiple approaches provide strong evidence for physiological relevance, while discrepancies identify areas requiring further investigation.