Recombinant Neisseria meningitidis serogroup B Uncharacterized membrane protein NMB1645 (NMB1645)

What is NMB1645 and what are its structural characteristics?

NMB1645 is an uncharacterized membrane protein from Neisseria meningitidis serogroup B. The protein consists of 446 amino acids and contains multiple predicted transmembrane domains characteristic of integral membrane proteins. The complete amino acid sequence is known and begins with MLNPSRKLVELVRILDEGGFIFSGDPVQATE and continues through to RNALAECGAAWLEPDRAAQEGRLKDQ . Structural analysis suggests it contains hydrophobic regions consistent with membrane-spanning domains, particularly in segments containing high proportions of hydrophobic amino acids such as leucine, isoleucine, and phenylalanine .

How is recombinant NMB1645 typically produced for research applications?

Recombinant NMB1645 is typically produced in E. coli expression systems. The full-length protein (amino acids 1-446) is commonly expressed with an N-terminal histidine tag to facilitate purification. After expression, the protein is purified using affinity chromatography, typically resulting in preparations with greater than 90% purity as determined by SDS-PAGE . The purified protein is often supplied as a lyophilized powder in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 to maintain stability during storage and shipping .

What is the genomic context of NMB1645 within the N. meningitidis serogroup B genome?

The NMB1645 gene is located within the Neisseria meningitidis serogroup B genome. It is identified with UniProt ID Q9JYD0 . While complete genomic context information is limited in the provided search results, research into meningococcal genomics has revealed that membrane proteins like NMB1645 are often part of operons that encode components involved in similar cellular functions. The gene's location and potential operon structure would be important considerations for understanding its regulation and potential functional relationships with other meningococcal proteins.

What are the potential functional roles of NMB1645 in Neisseria meningitidis pathogenesis?

While NMB1645 remains functionally uncharacterized, its membrane localization suggests potential roles in bacterial-host interactions, nutrient acquisition, or signaling. Based on research with other meningococcal membrane proteins, NMB1645 may contribute to adhesion, invasion, or immune evasion processes. The protein's sequence contains regions that could be involved in protein-protein interactions or substrate binding . Comparative studies with other characterized membrane proteins in Neisseria species, such as those identified in cross-reactive studies between N. lactamica and N. meningitidis, may provide insights into possible functions .

How does NMB1645 compare to other membrane proteins being investigated as vaccine candidates?

Unlike well-studied meningococcal vaccine candidates such as PorB, TbpB, and factor H binding protein (fHbp/LP2086), NMB1645 remains largely uncharacterized as a potential vaccine target . Factor H binding protein has progressed to clinical trials, showing promising immunogenicity and safety profiles in children and adolescents . To evaluate NMB1645's potential as a vaccine candidate, researchers would need to assess its conservation across strains, surface accessibility, immunogenicity, and ability to induce bactericidal antibodies. Cross-reactive studies similar to those performed with N. lactamica proteins could determine if NMB1645 shares epitopes with proteins known to provide protection in animal models .

What protein-protein interactions might NMB1645 participate in, and how might these be experimentally determined?

Based on its membrane localization, NMB1645 likely interacts with other membrane or periplasmic proteins. While specific interacting partners for NMB1645 are not identified in the provided search results , several experimental approaches could be employed to discover these interactions:

• Pull-down assays using His-tagged recombinant NMB1645 as bait

• Bacterial two-hybrid systems

• Cross-linking studies followed by mass spectrometry

• Co-immunoprecipitation with anti-NMB1645 antibodies

Identifying interacting partners would provide critical insights into the protein's functional role in meningococcal biology and potentially identify novel therapeutic targets.

What are the optimal conditions for handling and storing recombinant NMB1645 protein?

For optimal handling and storage of recombinant NMB1645:

• Store lyophilized protein at -20°C/-80°C upon receipt

• 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

• Aliquot to avoid repeated freeze-thaw cycles, which can degrade the protein

• For short-term use, store working aliquots at 4°C for up to one week

• Avoid repeated freezing and thawing

Prior to opening, briefly centrifuge the vial to bring contents to the bottom. These storage conditions maximize protein stability and experimental reproducibility.

How can researchers verify the structural integrity and functional activity of purified recombinant NMB1645?

To verify structural integrity and functional activity of recombinant NMB1645, researchers could employ the following methods:

For functional assays, researchers would need to develop specific tests based on hypothesized functions, such as binding assays with potential ligands or interaction partners identified through bioinformatic analysis.

What approaches can be used to generate antibodies against NMB1645 for immunological studies?

Generating high-quality antibodies against membrane proteins like NMB1645 poses unique challenges due to their hydrophobicity and potential conformational epitopes. Researchers could employ these approaches:

• Immunization with full-length recombinant His-tagged NMB1645

• Immunization with synthetic peptides corresponding to predicted extracellular domains

• Immunization with recombinant fragments representing hydrophilic regions

• DNA immunization with an NMB1645 expression vector

For mouse immunizations, protocols similar to those used for N. lactamica proteins could be adapted, where immunization schedules included three doses administered at regular intervals . Resulting antisera should be characterized for:

• Antibody titer (ELISA)

• Specificity (Western blot)

• Cross-reactivity with native protein (immunofluorescence microscopy)

• Functional activity (bactericidal assays)

How can mass spectrometry be applied to study NMB1645 and its interactions?

Mass spectrometry offers powerful approaches for studying membrane proteins like NMB1645. Similar to studies with other Neisseria proteins, surface-enhanced laser-desorption ionization time-of-flight mass spectroscopy (SELDI-TOF MS) can be employed to identify cross-reactive proteins . Additional mass spectrometry applications include:

• Proteomic identification of post-translational modifications

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe protein dynamics and solvent accessibility

• Cross-linking mass spectrometry (XL-MS) to map protein-protein interaction interfaces

• Intact protein mass spectrometry to confirm protein integrity and modifications

These approaches could reveal structural insights and interaction networks involving NMB1645, contributing to a better understanding of its role in meningococcal biology.

What genomic and transcriptomic approaches would help understand NMB1645 expression and regulation?

To understand NMB1645 expression and regulation, researchers could employ these genomic and transcriptomic approaches:

Similar approaches have been used to study expression patterns of other membrane proteins in N. meningitidis, revealing condition-specific regulation patterns that could suggest functional roles.

How can structural biology techniques be adapted to study membrane proteins like NMB1645?

Structural characterization of membrane proteins presents significant challenges. For NMB1645, researchers could consider these approaches:

• X-ray crystallography:

  • Requires detergent solubilization or lipidic cubic phase crystallization

  • Potential use of antibody fragments to stabilize conformations

  • May require protein engineering to enhance crystallizability

• Cryo-electron microscopy:

  • Sample preparation in nanodiscs or detergent micelles

  • Single-particle analysis for structure determination

  • Potential for studying protein complexes in near-native environments

• Nuclear magnetic resonance (NMR):

  • Solution NMR for soluble domains

  • Solid-state NMR for membrane-embedded regions

  • Can provide dynamics information not accessible by other methods

• Computational structure prediction:

  • Homology modeling if structural homologs exist

  • Ab initio modeling using methods like AlphaFold

  • Molecular dynamics simulations to study conformational flexibility

Each approach has strengths and limitations, and a multi-technique strategy would likely provide the most comprehensive structural insights.

How might gene knockout or knockdown of NMB1645 be achieved in N. meningitidis?

Genetic manipulation of N. meningitidis requires specialized approaches due to its transformation requirements and genome organization. For NMB1645 knockout/knockdown, researchers could employ:

• Homologous recombination:

  • Design constructs with antibiotic resistance cassettes flanked by sequences homologous to regions upstream and downstream of NMB1645

  • Transform N. meningitidis with linearized constructs

  • Select transformants on antibiotic-containing media

  • Verify deletion by PCR and sequencing

• CRISPR-Cas9 system:

  • Design guide RNAs targeting NMB1645

  • Introduce Cas9 and guide RNA via plasmid transformation

  • Provide repair template for precise gene editing

  • Screen transformants for successful editing

• Conditional expression systems:

  • Replace native promoter with inducible promoter

  • Control expression levels with inducer concentration

  • Allows study of essential genes where complete knockout might be lethal

• Antisense RNA or CRISPR interference:

  • Express antisense RNA or dCas9 with guide RNAs

  • Achieve knockdown rather than knockout

  • Useful for studying gene dosage effects

Similar approaches have been successfully applied to other membrane proteins in N. meningitidis to study their functions.

What phenotypic assays would be most informative for characterizing the function of NMB1645?

Based on its membrane localization, several phenotypic assays could provide insights into NMB1645 function:

• Growth curve analysis under various conditions:

  • Nutrient limitation

  • Stress conditions (pH, temperature, oxidative stress)

  • Presence of host factors

• Adhesion and invasion assays:

  • Human epithelial and endothelial cell models

  • Primary cell cultures

  • Organoid systems

• Biofilm formation:

  • Static and flow chamber models

  • Confocal microscopy analysis

  • Biomass quantification

• Serum resistance:

  • Survival in normal human serum

  • Complement deposition analysis

  • Opsonophagocytosis assays

• Animal infection models:

  • Mouse bacteremia models

  • Transgenic mice expressing human factors

  • Competitive index experiments comparing wild-type and mutant strains

Comparing wild-type, NMB1645 knockout/knockdown, and complemented strains would provide evidence for specific functional roles.

How does the amino acid sequence of NMB1645 compare across different Neisseria species and strains?

Comparative sequence analysis of NMB1645 across Neisseria species could reveal evolutionary relationships and functional importance. While specific comparative data is not provided in the search results, a comprehensive analysis would include:

• Sequence alignment across:

  • Different N. meningitidis serogroups

  • Commensal Neisseria species (N. lactamica, N. sicca)

  • Other pathogenic Neisseria (N. gonorrhoeae)

• Analysis of:

  • Sequence conservation in transmembrane domains versus extracellular loops

  • Conservation of potential functional motifs

  • Evidence of positive or purifying selection

• Phylogenetic analysis to determine:

  • Evolutionary relationships

  • Potential horizontal gene transfer events

  • Species-specific adaptations

Such analysis could identify conserved regions suitable for vaccine development or therapeutics, similar to approaches used with other meningococcal proteins .

What are the most promising research directions for understanding NMB1645 function?

Based on current knowledge gaps, the most promising research directions include:

• Comprehensive structural characterization using a combination of experimental and computational approaches

• Identification of interacting partners through proteomics and targeted interaction studies

• Gene knockout studies coupled with phenotypic analysis under various conditions

• Expression analysis during different growth phases and infection-relevant conditions

• Immunological studies to assess surface exposure and immunogenicity

• Comparative genomics across meningococcal strains to assess conservation and variability

These approaches would provide complementary insights into NMB1645 function and potential as a therapeutic target or vaccine candidate.

How might further characterization of NMB1645 contribute to vaccine development efforts?

Further characterization of NMB1645 could contribute to meningococcal vaccine development in several ways:

• Assessment as a direct vaccine antigen:

  • Determination of surface accessibility

  • Conservation analysis across strains

  • Immunogenicity studies

  • Protection in animal models

• Understanding of host-pathogen interactions:

  • Identification of critical interactions with host factors

  • Discovery of vulnerabilities that could be targeted

  • Insights into immune evasion mechanisms

Studies with other meningococcal proteins have shown that uncharacterized membrane proteins can emerge as promising vaccine candidates after thorough investigation . The approach of using N. lactamica proteins to provide cross-protection against N. meningitidis could be informative if NMB1645 has homologs in commensal species .

What techniques from systems biology could be applied to place NMB1645 in the broader context of meningococcal biology?

Systems biology approaches would help contextualize NMB1645 within meningococcal biology:

• Multi-omics integration:

  • Combine proteomics, transcriptomics, and metabolomics data

  • Identify co-regulated genes and proteins

  • Map to metabolic pathways and cellular processes

• Protein-protein interaction networks:

  • Generate comprehensive interactome maps

  • Identify functional modules and complexes

  • Predict functions based on interaction partners

• Flux balance analysis:

  • Incorporate membrane transporters into metabolic models

  • Predict effects of NMB1645 perturbation on cellular metabolism

  • Identify essential pathways linked to membrane function

• Comparative systems analysis:

  • Compare system-level data between pathogenic and commensal Neisseria

  • Identify pathogen-specific network features

  • Discover potential drug targets with minimal impact on commensal species