fkpA Antibody

What is FkpA and why is it significant in protein folding research?

FkpA is a periplasmic peptidyl-prolyl cis-trans isomerase (PPIase) that exhibits substantial chaperone activity in E. coli. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, facilitating proper protein folding . The significance of FkpA in research stems from its ability to increase solubility and decrease aggregation of various unfolded or misfolded proteins, particularly in challenging experimental conditions . FkpA has been demonstrated to improve the yields of recombinantly expressed proteins when co-expressed in the same system, making it valuable for protein expression and folding studies .

Though not essential for cell survival due to the redundant and functionally overlapping nature of the OMP biogenesis chaperone network, FkpA serves as a potent stress-response chaperone that is upregulated by the E. coli sigma E stress response . This makes it an important subject for understanding cellular adaptation mechanisms to stress conditions that increase the risk of protein misfolding.

What types of FkpA antibodies are available for research, and what epitopes do they typically recognize?

Commercial FkpA antibodies are available as polyclonal preparations that recognize specific amino acid sequences of the protein. For example, one commercially available antibody (ABIN7140383) is a rabbit polyclonal that specifically recognizes amino acids 21-268 of the FkpA protein from Aeromonas species . These antibodies are typically available in unconjugated forms for flexibility in experimental applications, though some may be available with biotin or other conjugates for specific detection methods .

The immunogen used for generating these antibodies often consists of recombinant FkpA protein or specific fragments. For instance, the ABIN7140383 antibody was raised against recombinant Aeromonas hydrophila FKBP-type peptidyl-prolyl cis-trans isomerase FkpA protein (amino acids 21-268) . The specificity for this region allows researchers to detect FkpA in various experimental contexts.

How is FkpA structurally organized, and what implications does this have for antibody epitope selection?

FkpA has a distinct structural organization consisting of two main domains connected by a long, flexible α-helix. The N-terminal domain contains the chaperone activity, while the C-terminal domain possesses the PPIase activity . This structural arrangement allows the two C-terminal domains to move independently of each other, which is important for its function .

The protein also contains unstructured tails at both the N- and C-termini that have not been ascribed any functional importance . When selecting or developing antibodies against FkpA, researchers should consider this domain organization. Antibodies targeting the N-terminal domain may be more useful for studying chaperone functions, while those recognizing the C-terminal domain would be better suited for investigating PPIase activity. The flexible connecting α-helix might present challenges for epitope recognition due to its dynamic nature.

How can FkpA antibodies be used to study protein folding mechanisms in the bacterial periplasm?

FkpA antibodies provide valuable tools for investigating protein folding mechanisms in the bacterial periplasm through several methodological approaches:

• Co-immunoprecipitation studies: Researchers can use FkpA antibodies to isolate FkpA-client protein complexes from bacterial lysates, allowing identification of proteins that interact with FkpA during folding processes . This approach has revealed that FkpA binds to various outer membrane proteins (OMPs) such as OmpA, OmpX, and BamA in their unfolded states .

• Localization studies: Immunofluorescence microscopy with FkpA antibodies can determine the spatial distribution of FkpA in bacteria under different stress conditions, revealing how its localization changes when protein folding stress increases in the periplasm.

• Quantification of expression levels: Western blotting with FkpA antibodies can quantify how FkpA expression changes in response to various stress conditions. This is particularly relevant since FkpA is upregulated by the E. coli sigma E stress response , making it a potential marker for periplasmic folding stress.

These approaches allow researchers to unravel the complex chaperone networks involved in periplasmic protein folding and to understand FkpA's contribution to maintaining protein homeostasis in this compartment.

What insights have been gained about OMP biogenesis using FkpA antibodies?

Studies utilizing FkpA antibodies have provided several key insights into OMP biogenesis:

• Functional redundancy in chaperone networks: FkpA antibody-based depletion studies have helped establish that FkpA can rescue the lethal phenotype of ΔsurA Δskp double mutants at elevated temperatures, demonstrating functional redundancy in the periplasmic chaperone network .

• Distinct folding trajectories: Immunological detection of FkpA-bound intermediates has revealed that FkpA binding to unfolded OMPs affects their folding trajectory in ways distinct from other periplasmic chaperones like SurA or Skp . Specifically, FkpA increases the total folded population of OMPs like OmpA, OmpX, and BamA, while reducing the rate of OmpA and OmpX folding .

• Client specificity: Immunoprecipitation with FkpA antibodies has helped identify that FkpA binds various OMP clients and prevents their aggregation, as demonstrated in sedimentation velocity analytical ultracentrifugation experiments .

These insights contribute to our understanding of how bacteria maintain OMP homeostasis and respond to periplasmic stress conditions.

How can researchers use FkpA antibodies in recombinant protein production optimization studies?

FkpA antibodies serve as essential tools in optimization studies for recombinant protein production through several methodological approaches:

• Validation of co-expression systems: When FkpA is co-expressed to enhance solubility of difficult-to-express proteins, antibodies can confirm FkpA expression levels through Western blotting, ensuring that sufficient chaperone is present in the system .

• Quantitative correlation studies: By quantifying both FkpA levels (using anti-FkpA antibodies) and target protein yields in various expression conditions, researchers can establish optimal FkpA:target protein ratios for maximum soluble protein production . This approach has been particularly valuable in optimizing the expression of engineered antibody fragments such as single-chain antibodies (scAbs) and Fab fragments .

• Subcellular localization: Antibodies against FkpA can determine whether FkpA is correctly localized to facilitate proper folding. This is especially relevant when using modified forms of FkpA, such as cytFkpA (lacking its signal sequence), which is expressed in the E. coli cytosol rather than the periplasm .

These applications have demonstrated that FkpA can substantially improve the soluble and functional expression of antibody fragments when either fused to the target protein or co-expressed in the same system .

What are the optimal conditions for using FkpA antibodies in Western blotting applications?

For optimal Western blotting with FkpA antibodies, the following methodological considerations should be implemented:

• Sample preparation: Cell lysates should be prepared with care to preserve FkpA's native structure. For periplasmic proteins like FkpA, osmotic shock extraction can provide enriched samples with reduced contamination from cytoplasmic proteins.

• Dilution ratios: Commercial FkpA antibodies typically perform optimally at dilutions between 1:1000 and 1:5000 for Western blotting applications . Researchers should perform dilution series to determine the optimal concentration for their specific experimental conditions.

• Detection systems: While either colorimetric or chemiluminescence detection can be used, enhanced chemiluminescence (ECL) systems often provide better sensitivity for detecting FkpA, particularly when studying its expression under stress conditions where levels may vary.

• Controls: Proper positive controls (purified recombinant FkpA) and negative controls (lysates from FkpA knockout strains) should be included to validate antibody specificity.

• Storage and handling: FkpA antibodies should be stored at -20°C or -80°C, and repeated freeze-thaw cycles should be avoided to maintain antibody activity .

Following these methodological guidelines ensures reliable and reproducible detection of FkpA in Western blotting experiments.

What experimental approaches can be used to study FkpA interactions with client proteins using FkpA antibodies?

Several sophisticated experimental approaches can be employed to study FkpA interactions with client proteins:

• Co-immunoprecipitation (Co-IP): FkpA antibodies can be used to pull down FkpA along with bound client proteins. This approach has revealed interactions between FkpA and unfolded OMPs such as OmpA 171, OmpX, and BamA . The methodology involves:

  • Cross-linking protein complexes with mild fixatives if interactions are transient

  • Immunoprecipitation with FkpA antibodies

  • SDS-PAGE separation and identification of co-precipitated proteins by mass spectrometry

• Surface Plasmon Resonance (SPR): Immobilized FkpA antibodies can be used to capture FkpA on SPR chips, followed by flowing potential client proteins to measure binding kinetics and affinities in real-time.

• Proximity Ligation Assays: This technique can detect FkpA-client interactions in situ with high sensitivity by using FkpA antibodies in combination with antibodies against potential client proteins.

• Sedimentation Analysis: As demonstrated in research, FkpA binding to client proteins can be analyzed through sedimentation velocity analytical ultracentrifugation (SV-AUC), with FkpA antibodies used to detect the chaperone in different fractions .

These approaches provide complementary information about the specificity, strength, and functional consequences of FkpA interactions with its client proteins.

What considerations should be made when using FkpA antibodies in ELISA applications?

When designing ELISA experiments with FkpA antibodies, researchers should consider the following methodological factors:

• Coating conditions: For direct ELISA, recombinant FkpA should be coated at 1-5 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C to ensure optimal antigen presentation.

• Blocking agents: BSA (bovine serum albumin) at 1-3% concentration is commonly used and appropriate for FkpA antibody applications, though optimization may be required for specific experimental setups .

• Antibody dilution: FkpA antibodies typically perform well at dilutions similar to those used in Western blotting (1:1000-1:5000), but each application may require optimization .

• Detection systems: Horseradish peroxidase (HRP) conjugated secondary antibodies with TMB (3,3',5,5'-tetramethylbenzidine) substrate provide sensitive detection for FkpA-based ELISAs .

• Controls: Include purified FkpA as a positive control and irrelevant proteins of similar size and abundance as negative controls to ensure specificity.

• Sandwich ELISA considerations: When designing sandwich ELISAs, ensure that the capture and detection antibodies recognize different epitopes on FkpA to prevent competitive binding.

These methodological considerations will help researchers develop robust ELISA protocols for various FkpA-related research applications.

How can researchers assess the impact of FkpA on OMP folding trajectories using antibodies?

Assessing FkpA's impact on OMP folding trajectories requires sophisticated experimental approaches:

To implement these methodologies, researchers can use FkpA antibodies to:

• Validate FkpA presence in folding reactions

• Immunoprecipitate FkpA-OMP complexes at different time points during folding

• Detect the distribution of FkpA across various fractions in sedimentation experiments

This multi-faceted approach has revealed that FkpA enhances membrane protein folding through an extensive binding interface, representing the first study to demonstrate a direct impact of FkpA binding and chaperoning on uOMP folding trajectories .

What methodological approaches can resolve contradictory findings regarding FkpA's dual chaperone and PPIase activities?

To resolve contradictions regarding FkpA's dual functions, researchers can employ several sophisticated methodological approaches using FkpA antibodies:

• Domain-specific antibody studies: Developing antibodies that specifically recognize either the N-terminal chaperone domain or the C-terminal PPIase domain allows selective inhibition of each function independently. This approach can determine which activity is essential for specific clients or under particular stress conditions.

• Structure-function correlation using mutant variants: Researchers can generate FkpA mutants with impaired chaperone or PPIase activity, then use antibodies to:

  • Confirm proper expression and folding of the mutant proteins

  • Immunoprecipitate client complexes to determine if binding is affected

  • Compare the impact on client protein folding through folding assays

• Comparative analysis with other periplasmic folding factors: By using antibodies against FkpA, SurA, and Skp simultaneously, researchers can assess the relative contributions of these chaperones in various experimental conditions. This approach has already revealed that FkpA has unique effects on OMP folding that distinguish it from SurA and Skp .

• Time-resolved studies of client interaction: Using FkpA antibodies in pulse-chase experiments can determine whether the chaperone or isomerase activity is engaged first during client protein folding, helping to establish the sequence of events in FkpA-mediated protein folding.

These methodological approaches can help clarify the mechanistic relationship between FkpA's chaperone and PPIase activities, resolving apparent contradictions in the literature.

How can researchers investigate the potential of cytoplasmic FkpA (cytFkpA) for enhancing antibody fragment expression using immunological techniques?

Recent research has demonstrated that cytoplasmic expression of FkpA lacking its signal sequence (cytFkpA) can enhance the secretion of functional antibody fragments into the E. coli periplasm . To further investigate this phenomenon, researchers can employ several methodological approaches:

• Quantitative comparison studies: Using anti-FkpA antibodies in Western blot analysis to quantify expression levels of periplasmic FkpA versus cytFkpA, and correlating these with target protein yields to determine optimal expression conditions.

• Co-localization studies: Employing immunofluorescence microscopy with antibodies against both FkpA and the antibody fragment to determine whether cytFkpA and the antibody fragment interact in the cytoplasm before secretion.

• Pull-down assays: Utilizing FkpA antibodies to isolate cytFkpA and associated proteins from different cellular compartments to identify potential interaction partners that might explain the enhanced secretion efficiency.

• Time-course studies: Using pulse-chase experiments with immunoprecipitation to track the movement of antibody fragments from synthesis through secretion, comparing cells with and without cytFkpA expression.

• Phage display optimization: As demonstrated in research, co-expression of cytFkpA during phage library panning significantly increases the number of unique clones selected, as well as their functional expression levels and diversity . FkpA antibodies can be used to confirm successful expression of cytFkpA during these procedures.

These methodological approaches can help elucidate the mechanism by which cytFkpA enhances antibody fragment secretion, potentially leading to improved protocols for recombinant antibody production in E. coli.

How might FkpA antibodies be utilized in studying stress response pathways in bacteria?

FkpA antibodies offer powerful tools for investigating bacterial stress response mechanisms, particularly since FkpA is upregulated by the E. coli sigma E (σE) stress response . Methodological approaches for such studies include:

• Temporal expression profiling: Using FkpA antibodies in time-course Western blot analyses to monitor FkpA upregulation following exposure to various stressors (heat shock, antibiotic treatment, oxidative stress), providing insights into the kinetics of periplasmic stress responses.

• Stress pathway delineation: Combining FkpA antibody detection with genetic approaches (knockouts of various stress response regulators) to map the regulatory networks controlling FkpA expression under different stress conditions.

• Cellular redistribution studies: Employing immunofluorescence microscopy with FkpA antibodies to track potential changes in FkpA localization during stress responses, which might reveal functional compartmentalization within the periplasm.

• Client profiling during stress: Using co-immunoprecipitation with FkpA antibodies to identify stress-specific client proteins that preferentially interact with FkpA under particular stress conditions.

These approaches can help establish FkpA as a marker for specific types of periplasmic stress and provide insights into bacterial adaptation mechanisms to adverse environmental conditions.

What methodological considerations should be addressed when developing improved FkpA antibodies for research applications?

Development of next-generation FkpA antibodies should consider several methodological factors to enhance their research utility:

• Epitope selection optimization: Carefully selecting epitopes that:

  • Are conserved across species if broad reactivity is desired

  • Are unique to specific bacterial species if species-specific detection is needed

  • Target functional domains to potentially block either chaperone or PPIase activity selectively

  • Remain accessible in native protein conformations for applications like immunoprecipitation

• Cross-reactivity assessment: Comprehensive validation against:

  • FkpA from different bacterial species

  • Related PPIases (like SlyD, PpiD, and other FKBPs)

  • Proteins with similar structural motifs

• Functional impact evaluation: Testing whether antibody binding affects:

  • Chaperone activity in folding assays

  • PPIase activity in isomerization assays

  • Client protein binding capacity

• Application-specific optimization:

  • For Western blotting: Optimizing for denatured epitope recognition

  • For immunoprecipitation: Focusing on native conformation recognition

  • For ELISA: Balancing sensitivity and specificity requirements

Addressing these methodological considerations during antibody development will yield more versatile and powerful tools for FkpA research across various experimental contexts.