Recombinant Haemophilus influenzae Uncharacterized protein HI_0219 (HI_0219)

What is HI_0219 and what organism does it come from?

HI_0219 is an uncharacterized protein derived from the gram-negative bacterium Haemophilus influenzae. It consists of 213 amino acids in its full-length form. As indicated by its designation as "uncharacterized," the specific biological function and structural characteristics of this protein remain largely unknown and represent an area requiring further investigation . Haemophilus influenzae is a respiratory pathogen that can cause various infections, including pneumonia, bacteremia, and meningitis, making proteins involved in its pathogenicity of significant research interest.

What expression systems are most effective for producing recombinant HI_0219?

Based on available data, E. coli appears to be the primary expression system used for producing recombinant HI_0219. The documented approach uses a His-tag for purification purposes . For researchers seeking to optimize expression, consider the following methodology:

• Select an E. coli strain optimized for recombinant protein expression (BL21(DE3), Rosetta, or SHuffle for proteins requiring disulfide bonds)

• Design expression constructs with appropriate promoters (T7 or tac)

• Optimize induction conditions (IPTG concentration, temperature, and duration)

• Test various growth media formulations (LB, TB, or auto-induction media)

• Consider codon optimization if expression levels are suboptimal

Alternative expression systems that might be explored include yeast (Pichia pastoris), insect cells (baculovirus expression system), or cell-free expression systems if E. coli expression proves challenging.

What purification strategies work best for recombinant HI_0219?

For His-tagged HI_0219, immobilized metal affinity chromatography (IMAC) represents the primary purification approach . A comprehensive purification protocol would typically include:

• Cell lysis using sonication or high-pressure homogenization in a buffer containing protease inhibitors

• Clarification by centrifugation (20,000 × g, 30 min, 4°C)

• IMAC purification using Ni-NTA or Co-based resins with an imidazole gradient

• Secondary purification via size exclusion chromatography (SEC) or ion exchange chromatography

• Buffer exchange to a stabilizing formulation (typically phosphate or Tris-based buffers with 100-150 mM NaCl)

Quality assessment via SDS-PAGE, Western blotting, and possibly mass spectrometry should be performed to verify purity and integrity of the purified protein.

What approaches can be used to characterize the function of HI_0219?

Elucidating the function of an uncharacterized protein like HI_0219 requires a multi-faceted approach:

• Sequence-based analysis: Apply bioinformatics tools including BLAST, PFAM, and INTERPRO to identify conserved domains or sequence similarities with characterized proteins. Structural prediction algorithms (AlphaFold, I-TASSER) can generate hypothetical models to guide functional studies.

• Genetic context analysis: Examine the genomic neighborhood of HI_0219 for clues about functional associations. Genes in the same operon often participate in related biological processes.

• Phenotypic studies: Generate knockout or knockdown strains of H. influenzae lacking functional HI_0219 and characterize phenotypic changes in growth, morphology, stress resistance, and virulence.

• Protein interaction studies: Employ yeast two-hybrid, pull-down assays, or proximity labeling techniques to identify protein binding partners, which may suggest functional pathways.

• Biochemical assays: Screen for potential enzymatic activities using substrate panels and activity-based protein profiling.

These methodologies should be employed iteratively, with results from one approach informing the design of subsequent experiments.

How can structural analysis inform functional hypotheses for HI_0219?

Structural characterization of HI_0219 can provide significant insights into its potential function:

• X-ray crystallography preparation: Optimize protein concentration (typically 5-20 mg/ml), buffer conditions, and screening of crystallization conditions using commercial kits. Consider surface entropy reduction mutations if crystallization proves challenging.

• NMR spectroscopy approach: For smaller domains of HI_0219, prepare isotopically labeled (15N, 13C) samples for structural determination in solution.

• Cryo-EM methodology: For larger complexes involving HI_0219, single-particle cryo-EM may be appropriate, requiring optimization of grid preparation and imaging conditions.

• Structure-based function prediction: Once structural data is obtained, use tools like DALI, COFACTOR, or COACH to identify structural homologs with known functions. Examine potential active sites, binding pockets, or interaction interfaces.

• Molecular dynamics simulations: Investigate conformational flexibility and potential binding modes with predicted interaction partners or substrates.

Structural features such as catalytic triads, nucleotide-binding motifs, or membrane-interaction domains can substantially narrow the range of possible functions for further experimental validation.

What computational methods can predict potential functions of HI_0219?

Several computational approaches can generate testable hypotheses about HI_0219 function:

• Homology-based methods: Beyond standard BLAST searches, employ Position-Specific Iterative BLAST (PSI-BLAST) and Hidden Markov Models (HMMs) to detect remote homologies that might not be apparent with standard sequence comparison tools.

• Structural prediction and comparison: Use AlphaFold2 or RoseTTAFold to generate structural models, then employ structure comparison algorithms like DALI or TM-align to identify structural homologs with known functions.

• Genomic context methods: Apply tools like STRING, GeCo or FunCoup to analyze conserved gene neighborhoods, gene fusion events, and phylogenetic profiles across bacterial species.

• Network-based inference: Use protein-protein interaction networks and pathway analysis to predict functional associations based on the "guilt by association" principle.

• Machine learning approaches: Employ supervised learning algorithms trained on characterized proteins to predict functional features based on sequence characteristics, predicted structural elements, and evolutionary conservation patterns.

These computational predictions should be treated as hypotheses requiring experimental validation rather than definitive functional assignments.

What controls should be included when working with recombinant HI_0219?

Rigorous experimental design for HI_0219 studies should include the following controls:

• Expression controls:

  • Empty vector control to distinguish host cell background

  • Inactive mutant version of HI_0219 (if catalytic residues are predicted)

  • Well-characterized protein expressed under identical conditions

• Purification controls:

  • Analysis of both soluble and insoluble fractions

  • Sequential sampling during purification steps

  • Mass spectrometry verification of protein identity

• Functional assay controls:

  • Positive controls using proteins with known activity in the assay system

  • Negative controls including heat-denatured HI_0219

  • Buffer-only and tag-only controls to assess background signals

• Interaction study controls:

  • Non-specific binding controls (e.g., GST or His-tag alone)

  • Competition assays with unlabeled protein

  • Reciprocal co-immunoprecipitation to confirm interactions

These controls help distinguish genuine biological activities from artifacts and ensure reproducibility across different experimental setups.

How can subcellular localization of HI_0219 be determined in Haemophilus influenzae?

Determining the subcellular localization of HI_0219 in its native context requires complementary approaches:

• Computational prediction:

  • Use tools like PSORTb, CELLO, or SignalP to predict subcellular targeting

  • Analyze for transmembrane domains using TMHMM or Phobius

  • Identify potential localization signals with specialized algorithms

• Fractionation studies:

  • Perform differential centrifugation to separate cytoplasmic, membrane, and periplasmic fractions

  • Use extraction methods with detergents of increasing strength to distinguish peripheral from integral membrane proteins

  • Verify fraction purity with known marker proteins for each compartment

• Immunolocalization:

  • Generate specific antibodies against HI_0219 or use epitope tags

  • Perform immunofluorescence microscopy with appropriate fixation methods

  • Consider immuno-electron microscopy for higher-resolution localization

• Reporter fusions:

  • Create translational fusions with fluorescent proteins like GFP

  • Use split-GFP systems for membrane protein topology studies

  • Validate with complementary approaches to ensure fusion proteins retain native localization

Localization data can provide significant clues about potential functions, as different subcellular compartments are associated with distinct biological processes.

What techniques are suitable for studying HI_0219 in a bacterial infection model?

To evaluate the potential role of HI_0219 in pathogenesis, consider these methodological approaches:

• Genetic manipulation strategies:

  • Create clean deletion mutants using allelic exchange

  • Develop conditional expression systems if HI_0219 is essential

  • Complement mutants with wild-type and mutated versions for phenotype rescue

• In vitro infection models:

  • Compare wild-type and HI_0219-deficient strains in adhesion to respiratory epithelial cells

  • Assess intracellular survival in macrophages or neutrophils

  • Measure biofilm formation capabilities under various conditions

• Animal models:

  • Utilize established mouse models of H. influenzae infection

  • Monitor bacterial loads in different tissues

  • Assess host immune responses through cytokine profiling and immune cell recruitment

• Transcriptomic approaches:

  • Perform RNA-seq comparing wild-type and HI_0219 mutant strains during infection

  • Identify differentially expressed genes to place HI_0219 in regulatory networks

  • Validate key findings with qRT-PCR and protein-level analyses

• Imaging techniques:

  • Apply two-photon microscopy to visualize bacterial behavior in vivo

  • Use reporter strains with fluorescent or luminescent markers to track infection dynamics

  • Consider intravital microscopy for real-time monitoring of host-pathogen interactions

These approaches can collectively reveal whether HI_0219 contributes to virulence or other aspects of H. influenzae pathophysiology.

How can protein-protein interactions of HI_0219 be comprehensively mapped?

Mapping the interactome of HI_0219 requires multiple complementary approaches:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged HI_0219 in H. influenzae or relevant host cells

  • Perform pull-down under native conditions

  • Identify co-purifying proteins via liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Filter against common contaminants using CRAPome or similar databases

• Proximity-dependent labeling:

  • Create fusions with BioID, TurboID, or APEX2 enzymes

  • Express in relevant biological context and activate labeling

  • Identify labeled proteins via streptavidin purification and MS

  • Validate key interactions with orthogonal methods

• Yeast two-hybrid screening:

  • Construct bait plasmids with HI_0219 fused to DNA-binding domains

  • Screen against prey libraries from H. influenzae

  • Confirm interactions via multiple selection markers

  • Test for direct interaction using purified proteins

• Protein complementation assays:

  • Split-reporter systems (luciferase, fluorescent proteins)

  • FRET or BRET for dynamic interaction studies

  • Mammalian two-hybrid for validation in eukaryotic contexts

• Crosslinking mass spectrometry:

  • Apply chemical crosslinkers to stabilize transient interactions

  • Identify crosslinked peptides via specialized MS workflows

  • Generate interaction maps with distance constraints

Computational analysis using clustering algorithms and network visualization tools can help identify functional modules and prioritize interactions for validation.

What approaches can resolve contradictory findings about HI_0219 function?

When faced with contradictory data about HI_0219 function, apply this systematic methodology:

• Critical evaluation of experimental conditions:

  • Compare protein preparation methods (tags, purification protocols)

  • Assess buffer compositions, pH, temperature, and ionic strength

  • Evaluate differences in cell types or bacterial strains used

• Independent validation:

  • Reproduce key experiments in different laboratories

  • Use orthogonal techniques to test the same hypothesis

  • Develop quantitative assays with appropriate statistical power

• Structural and conformational analysis:

  • Investigate if HI_0219 adopts multiple conformations

  • Assess oligomerization states under different conditions

  • Determine if post-translational modifications affect function

• Context-dependent functionality:

  • Test function under different physiological conditions

  • Investigate potential cofactor or binding partner requirements

  • Assess temporal regulation during different growth phases

• Integrated data analysis:

  • Apply Bayesian approaches to weigh evidence from different sources

  • Use meta-analysis techniques where appropriate

  • Consider mathematical modeling to reconcile apparently contradictory observations

This systematic approach can help determine whether contradictions reflect technical artifacts, context-dependent functions, or multiple biological activities of HI_0219.

How should sequence homology data for HI_0219 be interpreted?

Interpreting sequence homology data for uncharacterized proteins like HI_0219 requires a nuanced approach:

This interpretative framework helps avoid both over-interpretation (assuming functional similarity with distant homologs) and under-utilization of evolutionary insights.

What databases and resources are most valuable for comparative analysis of HI_0219?

For comprehensive analysis of HI_0219, researchers should utilize these databases and resources:

• Sequence databases and search tools:

  • UniProt/Swiss-Prot for manually curated protein information

  • RefSeq for comprehensive genomic context

  • HMMER web server for sensitive homology detection using profile HMMs

• Structural databases and prediction servers:

  • PDB for experimentally determined structures of homologs

  • AlphaFold DB for predicted structures

  • SWISS-MODEL for homology modeling

  • MobiDB for intrinsic disorder prediction

• Functional annotation resources:

  • InterPro for integrated domain and feature detection

  • KEGG for metabolic pathway mapping

  • GO (Gene Ontology) for standardized functional descriptions

  • BRENDA for enzyme function information

• Haemophilus-specific resources:

  • PATRIC for comparative genomics of bacterial pathogens

  • PortEco for model organism data from related species

  • Bacterial Microarray/RNA-seq databases for expression data

• Integrated analysis platforms:

  • DAVID for functional annotation clustering

  • STRING for protein-protein interaction networks

  • PAINT for phylogenetic-based function prediction

These resources should be used iteratively, with results from one analysis informing searches in other databases to build a comprehensive understanding of potential HI_0219 functions.

How might understanding HI_0219 function contribute to Haemophilus influenzae pathogenesis research?

Elucidating the function of HI_0219 could advance H. influenzae pathogenesis research in several ways:

• Virulence mechanism identification:

  • If HI_0219 participates in host-pathogen interactions, it may reveal novel virulence mechanisms

  • Understanding its regulation during infection could identify environmental triggers of pathogenicity

  • Potential involvement in biofilm formation or persistence mechanisms

• Therapeutic target assessment:

  • Evaluation as a potential drug target if essential for bacterial survival or virulence

  • Structure-based drug design opportunities if atomic resolution structures become available

  • Possibility of targeting conserved functions across multiple bacterial pathogens

• Diagnostic applications:

  • Development of detection methods if HI_0219 is expressed during specific infection stages

  • Potential serological marker if immunogenic in humans

  • Possible use in molecular typing of clinical isolates

• Evolutionary insights:

  • Understanding of how H. influenzae has adapted to its ecological niche

  • Comparison with homologs in other pathogens to identify convergent virulence strategies

  • Analysis of selection pressures shaping pathogen evolution

These contributions would extend beyond basic scientific understanding to potential clinical applications in diagnosis, treatment, or prevention of H. influenzae infections.

What methods can assess the potential of HI_0219 as a therapeutic target?

Evaluating HI_0219 as a therapeutic target requires a systematic approach:

• Target validation studies:

  • Determine essentiality using conditional knockouts or CRISPRi

  • Assess virulence contribution in relevant animal models

  • Evaluate conservation across clinical isolates to predict resistance development potential

• Druggability assessment:

  • Analyze structural features for potential binding pockets

  • Perform computational pocket analysis (fpocket, SiteMap)

  • Consider protein-protein interaction interfaces as potential targets

• High-throughput screening:

  • Develop functional assays amenable to screening

  • Design fragment-based screening approaches

  • Consider phenotypic screens in whole-cell contexts

• Structure-based drug design:

  • Obtain high-resolution structures of HI_0219

  • Perform in silico screening for potential inhibitors

  • Apply molecular dynamics to evaluate binding stability

• Lead optimization strategies:

  • Establish structure-activity relationships

  • Assess pharmacokinetic properties of lead compounds

  • Determine selectivity against human homologs or microbiome species

This multifaceted evaluation process can determine whether HI_0219 represents a viable therapeutic target worthy of significant drug development investment.

What are the specifications for commercially available recombinant HI_0219?

Available recombinant HI_0219 protein has the following specifications:

This commercially available recombinant protein is expressed in E. coli with a His-tag to facilitate purification and detection in experimental applications .