Recombinant Klebsiella pneumoniae subsp. pneumoniae UPF0266 membrane protein KPN78578_23010 (KPN78578_23010)

What is the KPN78578_23010 protein and what are its basic structural features?

KPN78578_23010 is a UPF0266 family membrane protein from Klebsiella pneumoniae subsp. pneumoniae. It is a full-length protein consisting of 152 amino acids with a molecular structure characteristic of membrane proteins. The protein features transmembrane domains that allow its integration into the bacterial membrane. The complete amino acid sequence is: MTFTDLVIILFILALLAYAIYDQFIMPRRNGPVLLAIPLLRRSRVDGMIFVGLTAILIYNNITQHGTAITTWLLSVLALMGLYLFWIRTPKIIFKPRGFFFANVWIEYQRIKEMNLSEDGVLVMQLEQRRLLIRVRNIDDLEKIYKLLITTQ .

How is recombinant KPN78578_23010 protein typically expressed and purified?

Recombinant KPN78578_23010 is typically expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification. The protein spans amino acids 1-152 of the native sequence. After expression, the protein is purified using affinity chromatography methods that exploit the His-tag, followed by additional purification steps to achieve greater than 90% purity as determined by SDS-PAGE analysis . The expression in E. coli allows for high yield production while maintaining the structural and functional properties necessary for research applications.

What are the recommended storage and handling conditions for this protein?

KPN78578_23010 recombinant protein is typically supplied as a lyophilized powder and should be stored at -20°C to -80°C upon receipt. Aliquoting is necessary for multiple use to prevent protein degradation from repeated freeze-thaw cycles. Working aliquots can be stored at 4°C for up to one week . The storage buffer consists of a Tris/PBS-based solution with 6% trehalose at pH 8.0, which helps maintain protein stability during storage. These conditions help preserve the structural integrity and functional activity of the protein for experimental applications.

What is the optimal protocol for reconstituting lyophilized KPN78578_23010 for experimental use?

For optimal reconstitution of lyophilized KPN78578_23010, first centrifuge the vial briefly to bring contents to the bottom. Reconstitute the protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. To enhance long-term stability, add glycerol to a final concentration of 5-50% (recommended default is 50%). After reconstitution, the solution should be aliquoted and stored at -20°C to -80°C for long-term use . This protocol minimizes protein aggregation and maximizes stability, ensuring consistent results across experiments.

How can researchers validate the structural integrity of KPN78578_23010 after reconstitution?

Validation of structural integrity can be performed through multiple complementary techniques. Primary assessment should include SDS-PAGE to confirm the expected molecular weight and purity (should be >90%) . Circular dichroism spectroscopy can evaluate secondary structure elements characteristic of membrane proteins. For more detailed structural analysis, limited proteolysis followed by mass spectrometry can verify the correct folding by identifying accessible cleavage sites. Functional assays specific to membrane proteins, such as liposome incorporation efficiency or membrane association tests, provide additional validation of proper protein conformation after reconstitution.

What experimental approaches are recommended for studying KPN78578_23010 interactions with other bacterial proteins?

To investigate KPN78578_23010 interactions with other bacterial proteins, researchers should employ a multi-technique approach. Pull-down assays utilizing the His-tag can capture protein complexes, while co-immunoprecipitation with specific antibodies against KPN78578_23010 can identify native interaction partners. Cross-linking studies using membrane-permeable crosslinkers followed by mass spectrometry analysis can capture transient interactions. For validation, bacterial two-hybrid systems adapted for membrane proteins or biolayer interferometry (BLI) with immobilized KPN78578_23010 can quantify binding kinetics. When designing these experiments, it's important to consider the membrane environment by using detergent micelles or nanodiscs to maintain native-like conditions.

What is currently known about the function of KPN78578_23010 in Klebsiella pneumoniae biology?

While the exact biological function of KPN78578_23010 remains to be fully elucidated, structural analysis indicates it belongs to the UPF0266 membrane protein family . Membrane localization suggests potential roles in transport, signaling, or maintenance of membrane integrity. Comparative genomic analysis with other Klebsiella strains reveals conservation of this protein, indicating fundamental importance in bacterial physiology. The protein's transmembrane domains suggest functions possibly related to substance transport or sensing environmental changes, though specific substrates or signals have not been definitively identified. This knowledge gap represents an important area for further research into Klebsiella pneumoniae biology.

How does KPN78578_23010 compare structurally and functionally to other membrane proteins in Klebsiella pneumoniae?

KPN78578_23010 differs structurally from better-characterized Klebsiella pneumoniae membrane proteins like OmpA and OmpK36, which are known to function in membrane stability and porin activity, respectively . While OmpA and OmpK36 have been extensively studied for their roles in virulence and have been utilized as vaccine candidates, the UPF0266 family protein KPN78578_23010 has a distinct amino acid sequence and predicted membrane topology . Unlike the beta-barrel structure typical of outer membrane proteins, KPN78578_23010's sequence suggests a different structural organization. Functional comparison is limited by the current lack of specific functional characterization for KPN78578_23010, highlighting an area requiring further investigation.

What potential role might KPN78578_23010 play in antimicrobial resistance mechanisms?

As a membrane protein, KPN78578_23010 could potentially contribute to antimicrobial resistance through several mechanisms, though direct evidence is currently limited. Membrane proteins can participate in resistance by altering membrane permeability, acting as efflux pump components, or modifying the bacterial cell envelope to reduce antibiotic penetration. The amino acid sequence of KPN78578_23010 (MTFTDLVIILFILALLAYAIYDQFIMPRRNGPVLLAIPLLRRSRVDGMIFVGLTAILIYNNITQHGTAITTWLLSVLALMGLYLFWIRTPKIIFKPRGFFFANVWIEYQRIKEMNLSEDGVLVMQLEQRRLLIRVRNIDDLEKIYKLLITTQ) could be analyzed for motifs associated with transporter or efflux functions. Investigation into expression levels of KPN78578_23010 in drug-resistant versus susceptible strains would provide valuable insights into its potential contribution to the significant antibiotic resistance issues associated with K. pneumoniae infections .

What are the major challenges in expressing and purifying KPN78578_23010, and how can they be addressed?

Expression and purification of membrane proteins like KPN78578_23010 present several challenges. The hydrophobic nature of membrane proteins often leads to aggregation, misfolding, or toxicity to the expression host. To address these issues, researchers should: (1) Optimize expression conditions including temperature (typically lowered to 16-25°C), inducer concentration, and duration; (2) Consider specialized E. coli strains designed for membrane protein expression; (3) Use fusion partners beyond the His-tag that enhance solubility; (4) Carefully select detergents for extraction that maintain native structure; and (5) Implement quality control steps including SEC-MALS to assess monodispersity. For KPN78578_23010 specifically, the relatively small size (152 amino acids) may facilitate expression compared to larger membrane proteins, but careful optimization of detergent conditions remains critical.

How can researchers design experiments to investigate potential immunogenic properties of KPN78578_23010?

To investigate the immunogenic properties of KPN78578_23010, researchers should design experiments that build on successful approaches used with other K. pneumoniae membrane proteins. Initial epitope mapping should be performed using computational tools to predict potential B-cell and T-cell epitopes within the KPN78578_23010 sequence. These predicted epitopes can then be synthesized and tested for immune recognition using sera from patients recovered from K. pneumoniae infections. For in vivo studies, purified KPN78578_23010 can be administered with appropriate adjuvants to mouse models, followed by evaluation of antibody titers, isotype distribution, and T-cell responses . Subsequent challenge studies would assess protective efficacy. Researchers should include appropriate controls such as other K. pneumoniae membrane proteins (e.g., OmpA or OmpK36) with established immunogenicity profiles for comparison .

What controls and validation steps are essential when studying protein-protein interactions involving KPN78578_23010?

When studying protein-protein interactions involving KPN78578_23010, several essential controls and validation steps must be implemented: (1) Include negative controls using non-specific proteins of similar size and charge characteristics; (2) Perform reverse pull-down experiments where potential interacting partners are tagged and used as bait; (3) Include competition assays with untagged protein to confirm specificity; (4) Validate interactions using at least two independent methods (e.g., pull-down followed by FRET analysis); (5) Perform dose-dependency tests to establish binding affinity ranges; and (6) Create targeted mutations in predicted interaction domains to confirm specific binding regions. For membrane proteins specifically, controls should include membrane fractions from bacteria not expressing KPN78578_23010 to account for non-specific membrane associations.

What is the potential of KPN78578_23010 as a target for vaccine or therapeutic development?

The potential of KPN78578_23010 as a vaccine or therapeutic target can be evaluated by examining precedents set by other K. pneumoniae membrane proteins. Recent research has demonstrated success with multi-epitope approaches using membrane proteins such as OmpA and OmpK36, which elicited protective immune responses in mouse models . For KPN78578_23010 to be considered a viable vaccine candidate, researchers would need to: (1) Determine the surface exposure and accessibility of regions of the protein; (2) Assess conservation across clinical isolates; (3) Evaluate immunogenicity in animal models; and (4) Demonstrate protective efficacy against challenge. Subunit vaccine approaches, similar to the r-AK36 model, could be adapted for KPN78578_23010 if immunogenic epitopes are identified . The growing threat of multidrug-resistant K. pneumoniae makes novel vaccine targets particularly valuable for future research .

How can molecular dynamics simulations contribute to understanding KPN78578_23010 structure-function relationships?

Molecular dynamics (MD) simulations can provide crucial insights into KPN78578_23010 structure-function relationships by modeling its behavior within a membrane environment. Starting with the amino acid sequence (MTFTDLVIILFILALLAYAIYDQFIMPRRNGPVLLAIPLLRRSRVDGMIFVGLTAILIYNNITQHGTAITTWLLSVLALMGLYLFWIRTPKIIFKPRGFFFANVWIEYQRIKEMNLSEDGVLVMQLEQRRLLIRVRNIDDLEKIYKLLITTQ) , researchers can: (1) Generate 3D structural predictions using AlphaFold or similar tools; (2) Embed the predicted structure in a lipid bilayer mimicking bacterial membranes; (3) Run extended simulations (100+ ns) to observe conformational dynamics; (4) Identify stable structural elements and flexible regions; and (5) Analyze potential binding pockets or channels. MD simulations have successfully been applied to study stability and interactions of other K. pneumoniae proteins with their receptors, as demonstrated in recent vaccine candidate research where 100 ns simulations revealed stable protein-receptor complexes .

What experimental approaches could elucidate the role of KPN78578_23010 in biofilm formation and virulence?

To investigate the potential role of KPN78578_23010 in biofilm formation and virulence, researchers should implement a comprehensive experimental strategy: (1) Generate knockout and overexpression strains of KPN78578_23010 in K. pneumoniae; (2) Assess changes in biofilm formation using crystal violet assays, confocal microscopy, and flow cell systems; (3) Evaluate virulence factor expression in these modified strains; (4) Conduct in vitro infection models using relevant cell lines; (5) Perform transcriptomic and proteomic analysis to identify pathways affected by KPN78578_23010 alteration; and (6) Use animal models to assess changes in colonization and virulence. Drawing parallels from other K. pneumoniae membrane proteins, researchers might consider testing whether antibodies against KPN78578_23010 demonstrate biofilm inhibition properties similar to those observed with anti-r-AK36 antibodies .