Recombinant Pseudomonas aeruginosa Protein fptB (fptB)

What is the basic structure and characteristics of Pseudomonas aeruginosa Protein fptB?

Protein fptB (UniProt ID: P42513) is a mature protein spanning amino acids 26-93 in Pseudomonas aeruginosa, with the amino acid sequence ASGYLLTRGLPLDDPLERLYAGLFGALGVGLLLLVGGLLARGPGNFAWRLGGSLLVLGLALWLLAGRG . Structural analysis suggests fptB contains multiple hydrophobic regions that likely contribute to membrane association, similar to other P. aeruginosa membrane proteins. The high prevalence of leucine (L) and glycine (G) residues within the sequence indicates potential flexibility in protein folding and membrane integration. Computational modeling suggests fptB adopts a predominantly alpha-helical secondary structure, which is common for bacterial membrane-associated proteins that interact with the bacterial envelope.

When working with recombinant versions, researchers should note that the addition of tags (commonly His-tags) may slightly alter the protein's physical properties but generally maintain functional characteristics . The protein has a predicted molecular weight of approximately 7.3 kDa (without tags), making it relatively small compared to other bacterial membrane-associated proteins. Understanding these basic structural characteristics is essential for designing appropriate expression, purification, and functional studies.

How can I optimize recombinant expression of fptB protein?

Optimizing recombinant expression of fptB requires careful consideration of expression systems, culture conditions, and purification strategies. The most common expression system for fptB is E. coli, which has been successfully used to produce the His-tagged recombinant protein . When designing expression vectors, consider codon optimization for E. coli to enhance translation efficiency, as P. aeruginosa has different codon usage patterns. Include appropriate signal sequences if targeting specific cellular compartments is desired.

Temperature modulation during induction is critical, with lower temperatures (16-25°C) often resulting in improved folding and solubility compared to standard 37°C expression. For membrane-associated proteins like fptB, expression in the presence of mild detergents or using specialized E. coli strains (such as C41/C43) may improve yield and solubility. Induction parameters, including IPTG concentration and duration, should be empirically optimized through small-scale expression trials before scaling up.

For purification, immobilized metal affinity chromatography (IMAC) using the His-tag is effective for initial capture, followed by size exclusion chromatography to achieve >90% purity as verified by SDS-PAGE . To maintain protein stability during and after purification, consider including glycerol (5-50%) in storage buffers, with 50% being commonly used for long-term storage at -20°C/-80°C . Avoid repeated freeze-thaw cycles, as they can significantly reduce protein activity and integrity.

What are the known functional roles of fptB in Pseudomonas aeruginosa biology?

While specific information about fptB's function is limited in the available literature, its structural features suggest roles potentially related to membrane integrity or transport processes in P. aeruginosa. The protein's hydrophobic regions indicate membrane association, which may contribute to cell envelope stability or selective permeability. Some membrane-associated proteins in P. aeruginosa play critical roles in virulence, antibiotic resistance, or biofilm formation, making fptB an intriguing target for investigation in these contexts.

P. aeruginosa employs numerous membrane proteins to facilitate pathogen-host interactions, including activation of pattern recognition receptors like Toll-like receptors (TLRs) . The bacterium's membrane proteins also contribute to its notorious antibiotic resistance through mechanisms like efflux pumps, altered membrane permeability, and biofilm formation . Understanding fptB's potential involvement in these processes requires comparative studies with other well-characterized membrane proteins like OprF, which has established roles in membrane integrity and immunogenicity .

Research investigating protein-protein interactions, knockout studies, and transcriptional analysis during different growth conditions or infection models would provide valuable insights into fptB's biological significance. Given P. aeruginosa's clinical importance as a multidrug-resistant pathogen, characterizing proteins like fptB may reveal new therapeutic targets or vaccine candidates.

How can I design experiments to investigate potential immunogenic properties of recombinant fptB?

Investigating the immunogenic properties of recombinant fptB requires a systematic approach similar to that used for other P. aeruginosa membrane proteins. Begin with in silico epitope prediction using algorithms specifically designed for bacterial antigens to identify potential B-cell and T-cell epitopes within the fptB sequence. These predictions can guide the design of immunization strategies and antibody detection assays.

For in vitro assessment, stimulate immune cells (such as dendritic cells or macrophages) with purified recombinant fptB and measure cytokine production, cell surface activation markers, and antigen presentation capabilities. Flow cytometry and ELISA-based assays are valuable tools for these assessments. Compare these responses to those elicited by known immunogenic proteins like OprF, which has been extensively studied for its immunogenic properties in P. aeruginosa vaccine development .

Animal immunization studies should follow established protocols like those used for OprF-LTB fusion proteins, where BALB/c mice and rabbits were immunized with the recombinant protein . Measure both humoral (antibody production) and cell-mediated immune responses through techniques such as ELISA, ELISpot, and flow cytometry. Consider investigating adjuvant effects by comparing fptB alone versus fptB fused with known immunomodulators like LTB, which has been shown to enhance immune responses to P. aeruginosa antigens . Protective efficacy can be assessed through challenge studies in appropriate animal models, such as burn wound infection models where P. aeruginosa is a common pathogen .

What strategies can overcome solubility and stability challenges with recombinant fptB?

Membrane-associated proteins like fptB often present significant solubility and stability challenges during recombinant expression and purification. A multi-faceted approach is necessary to address these issues. Consider fusion partners beyond simple affinity tags; solubility-enhancing partners such as SUMO, thioredoxin, or MBP can dramatically improve expression and solubility. These fusion partners can be removed post-purification using specific proteases if native protein is required.

The choice of detergents is critical for membrane protein stability. Screen a panel of detergents including mild non-ionic options (DDM, LDAO, OG) at concentrations above their critical micelle concentration. Alternative solubilization strategies include amphipols, nanodiscs, or styrene maleic acid lipid particles (SMALPs), which provide more native-like membrane environments and can enhance stability for functional and structural studies.

Buffer optimization should be systematic, testing different pH ranges (typically 6.0-8.5), salt concentrations (100-500 mM), and stabilizing additives such as glycerol (5-50%), trehalose (6% is used in standard formulations) , and reducing agents if cysteine residues are present. Protein stability can be quantitatively assessed using thermal shift assays (TSA/DSF) to identify optimal buffer conditions that maximize the protein's melting temperature.

For long-term storage, lyophilization has proven effective for fptB , but reconstitution protocols must be carefully developed and validated. When reconstituting lyophilized fptB, use deionized sterile water to achieve concentrations of 0.1-1.0 mg/mL, and consider adding glycerol to a final concentration of 50% for subsequent storage . Aliquoting is essential to avoid repeated freeze-thaw cycles, which significantly accelerate protein degradation.

How can I validate the structural integrity and functionality of purified recombinant fptB?

Validating structural integrity and functionality of purified recombinant fptB requires multiple complementary approaches. Basic validation begins with SDS-PAGE to confirm protein purity (>90% is considered acceptable for most applications) and Western blotting using anti-His antibodies or custom anti-fptB antibodies if available. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) provides information about oligomeric state and homogeneity.

Secondary structure analysis using circular dichroism (CD) spectroscopy can confirm the predicted alpha-helical content of fptB and monitor structural changes under different conditions. More detailed structural information can be obtained through techniques like nuclear magnetic resonance (NMR) spectroscopy, which is particularly suitable for smaller proteins like fptB (7.3 kDa).

Functional validation depends on the hypothesized role of fptB, but may include membrane association assays using liposomes or membrane mimetics, binding studies with potential interaction partners, or activity assays if enzymatic activity is predicted. For membrane proteins, reconstitution into proteoliposomes or nanodiscs followed by functional assays provides the most physiologically relevant assessment.

Stability assessment under various storage conditions is essential for experimental reproducibility. Samples stored under different conditions (temperature, buffer composition, duration) should be periodically analyzed using the methods described above to establish optimal storage protocols. For fptB specifically, avoiding repeated freeze-thaw cycles is recommended, and working aliquots should be stored at 4°C for no more than one week .

How might recombinant fptB contribute to vaccine development against Pseudomonas aeruginosa?

Recombinant fptB holds potential for vaccine development against P. aeruginosa, particularly when considered within the context of other successful outer membrane protein immunogens. The conserved nature of many membrane proteins across P. aeruginosa strains makes them attractive vaccine candidates. Similar to OprF, which has been studied extensively as a vaccine antigen, fptB could be evaluated both individually and as part of multi-antigen formulations to enhance protective immunity against P. aeruginosa infections .

Fusion protein strategies, particularly those incorporating known adjuvants like the B subunit of heat-labile enterotoxin (LTB), have shown promising results for enhancing immunogenicity. Research with OprF-LTB demonstrated significantly higher survival rates in immunized mice compared to controls when challenged with P. aeruginosa . Similar approaches could be applied to fptB, creating fusion proteins that enhance its immunogenicity and potentially provide cross-protection against multiple P. aeruginosa strains.

Evaluation of protective efficacy should follow established protocols in relevant animal models. For P. aeruginosa, burn wound infection models are particularly relevant, as demonstrated in studies with OprF where immunized animals showed significantly reduced bacterial loads in hepatic and splenic tissues following challenge . Both active immunization (with purified recombinant protein) and passive immunization (with antibodies raised against the protein) strategies should be assessed, as anti-OprF IgG has been shown to confer up to 75% survival in mice challenged with P. aeruginosa .

Advanced vaccine formulation considerations include delivery systems (such as liposomes or nanoparticles), adjuvant selection beyond protein-based adjuvants, and prime-boost strategies to maximize both antibody production and cell-mediated immunity. Comprehensive immune profiling, including assessment of T-cell responses, cytokine profiles, and antibody subclasses, will provide insights into the quality and potential protective mechanisms of the immune response elicited by fptB-based vaccine candidates.

What role might fptB play in Pseudomonas aeruginosa biofilm formation and how can this be studied?

Investigating fptB's potential role in P. aeruginosa biofilm formation requires sophisticated experimental approaches that bridge molecular and cellular analyses. P. aeruginosa biofilms represent a major clinical challenge, with up to 80% of human microbial infections being biofilm-associated . Initial investigations should include comparative expression analysis of fptB between planktonic and biofilm growth states using quantitative PCR and proteomics approaches to determine if fptB expression is regulated during biofilm development.

Gene knockout or knockdown studies using CRISPR-Cas9 or antisense RNA techniques would reveal phenotypic changes in biofilm formation capability when fptB expression is altered. Complementation studies with recombinant fptB would confirm specificity of any observed effects. Biofilm quantification should employ multiple methods including crystal violet staining, confocal laser scanning microscopy with fluorescent reporters, and biomass determination through dry weight measurement or protein quantification.

Advanced biofilm characterization techniques such as isothermal microcalorimetry can detect metabolic activity within biofilms with high sensitivity, as has been used for evaluating bacteriophage efficacy against P. aeruginosa biofilms . Additionally, quantitative PCR can be used to assess bacterial abundance within biofilms following various treatments or genetic modifications . These methods provide complementary information about biofilm structure, composition, and metabolic state.

If fptB is found to influence biofilm formation, mechanistic studies should follow to determine if this occurs through effects on cell surface properties, extracellular matrix production, quorum sensing systems, or other pathways known to regulate biofilm development in P. aeruginosa. Recombinant fptB could also be evaluated as a potential anti-biofilm agent if it competitively inhibits native protein function or disrupts important protein-protein interactions required for biofilm integrity.

What are common troubleshooting steps for low yield or inactive recombinant fptB?

Low yield or inactivity of recombinant fptB can stem from multiple factors requiring systematic troubleshooting. Expression issues often arise from toxicity to the host cells, particularly for membrane proteins. If growth inhibition is observed upon induction, consider using tightly regulated expression systems, lower inducer concentrations, or specialized E. coli strains designed for toxic protein expression such as C41(DE3) or BL21(DE3)pLysS. Codon optimization for E. coli can also address translational limitations when expressing P. aeruginosa proteins.

Inclusion body formation is a common challenge with membrane proteins. While fptB can be purified from inclusion bodies through denaturation and refolding protocols, native-like folding is often better achieved through soluble expression. Lowering the induction temperature to 16-20°C, reducing inducer concentration, and co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) can significantly improve soluble yield. For membrane proteins like fptB, expression in the presence of mild detergents or phospholipids can facilitate proper folding.

Purification challenges include poor binding to affinity resins, which may indicate tag inaccessibility. Consider repositioning the His-tag from N-terminal to C-terminal or vice versa, or using dual affinity tags. If protein degradation occurs during purification, include protease inhibitors throughout the process and minimize processing time. For fptB specifically, maintaining appropriate buffer conditions is crucial, with Tris/PBS-based buffers at pH 8.0 containing 6% trehalose having been successfully used .

Activity loss can result from improper storage or handling. Avoid repeated freeze-thaw cycles as explicitly recommended for fptB , and store working aliquots at 4°C for no more than one week. For long-term storage, adding glycerol to 50% final concentration before freezing at -20°C/-80°C helps maintain structural integrity . If activity is still suboptimal, consider the possibility that additional cofactors or interaction partners present in the native P. aeruginosa environment may be required for full functionality.

How can I detect protein-protein interactions involving fptB in Pseudomonas aeruginosa?

Detecting protein-protein interactions involving fptB requires applying multiple complementary techniques to overcome the challenges associated with membrane proteins. Begin with in silico prediction tools specifically designed for bacterial protein interactions, which can generate hypotheses about potential interaction partners based on genomic context, co-expression data, and structural motifs. These predictions should guide subsequent experimental approaches.

Co-immunoprecipitation (Co-IP) using antibodies against fptB or its predicted partners is a standard approach but requires careful optimization for membrane proteins. Crosslinking prior to cell lysis can stabilize transient interactions. Chemical crosslinkers with different spacer arm lengths (such as DSP, DTSSP, or formaldehyde) should be tested to identify optimal conditions. Membrane solubilization requires careful detergent selection to maintain protein-protein interactions while effectively extracting membrane proteins.

Bacterial two-hybrid systems, particularly those adapted for membrane proteins such as BACTH (Bacterial Adenylate Cyclase Two-Hybrid), offer an in vivo approach to detect interactions. Split-reporter approaches like bimolecular fluorescence complementation (BiFC) can visualize interactions within bacterial cells. These genetic systems can be implemented in either E. coli or, preferably, in P. aeruginosa itself for more physiologically relevant results.

Advanced proteomic approaches provide unbiased identification of interaction networks. Proximity-dependent biotin identification (BioID) or APEX2-based proximity labeling, adapted for bacterial systems, allow identification of proteins in close proximity to fptB in living bacteria. Quantitative proteomics comparing wild-type to fptB-deficient strains can reveal altered protein complexes. For direct physical interaction assessment, recombinant fptB can be immobilized on biosensor surfaces (such as those used in surface plasmon resonance or bio-layer interferometry) to measure binding kinetics with purified candidate partners or P. aeruginosa lysates.

What emerging technologies are most promising for advancing research on fptB and similar bacterial membrane proteins?

Emerging technologies are revolutionizing the study of bacterial membrane proteins like fptB, offering unprecedented insights into structure, function, and potential therapeutic applications. Cryo-electron microscopy (cryo-EM) has transformed structural biology of membrane proteins, enabling determination of near-atomic resolution structures without crystallization. For smaller proteins like fptB (7.3 kDa), advances in microED (micro-electron diffraction) may be particularly valuable, as this technique has successfully resolved structures of small proteins that resist traditional structural approaches.

Artificial intelligence approaches, particularly AlphaFold2 and RoseTTAFold, are dramatically accelerating structural predictions of bacterial proteins, including those with limited homology to known structures. These computational models provide excellent starting points for rational experimental design and can predict protein-protein interactions with increasing accuracy. For membrane proteins specifically, specialized neural networks trained on membrane protein datasets offer improved prediction of transmembrane regions and protein orientation within the membrane.

Single-cell technologies applied to bacteria are revealing heterogeneity in protein expression and function that may be particularly relevant for membrane proteins involved in environmental adaptation or virulence. Single-cell RNA-seq and newer techniques like Ribo-seq at single-cell resolution can identify condition-specific expression patterns of fptB within P. aeruginosa populations. These approaches will be particularly valuable for understanding fptB expression during host-pathogen interactions or biofilm formation.