fkpA Antibody, Biotin conjugated

What is FkpA and why is it significant for researchers?

FkpA is a periplasmic protein in Escherichia coli that exhibits dual functionality as both a peptidyl-prolyl cis/trans isomerase (PPIase) and a molecular chaperone. Its significance lies in its role in protein folding and quality control in the bacterial periplasm.

The protein is a dimeric molecule with a V-shaped structure where each 245-residue subunit consists of two domains. The N-terminal domain contains three helices that intertwine with the other subunit to maintain the dimeric structure and provides chaperone activity. Meanwhile, the C-terminal domain belongs to the FK506-binding protein (FKBP) family and is responsible for PPIase activity .

FkpA has been shown to accelerate protein folding by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides . This dual functionality makes it an important target for studying bacterial stress responses, protein folding mechanisms, and potential antimicrobial strategies.

How does biotin conjugation enhance antibody applications in FkpA research?

Biotin conjugation creates a powerful tool for detecting and isolating FkpA from complex biological samples due to the following methodological advantages:

• High affinity binding: The extremely high affinity between biotin and streptavidin/avidin (Kd ≈ 10^-15 M) provides exceptional specificity and sensitivity .

• Signal amplification: Multiple detection systems can bind to each biotin molecule, amplifying signal strength in detection applications.

• Versatility in detection systems: Biotin-conjugated antibodies can be detected using various streptavidin/avidin conjugates (HRP, fluorophores, gold particles), enabling application across multiple platforms including ELISA, Western blot, and immunohistochemistry .

• Quantification capability: The biotin chromophore allows for spectrophotometric determination of both antibody concentration and average number of biotins per antibody molecule by measuring absorbance at A350 and A280 .

The conjugation procedure typically involves a simple process where the antibody reconstitutes a freeze-dried activator compound, which is then added to a biotin chromophore ligand. After a fixed incubation period, the conjugate is desalted to remove excess ligand and can be quantified spectrophotometrically .

What are the recommended storage and handling conditions for biotin-conjugated FkpA antibodies?

Optimal storage and handling of biotin-conjugated FkpA antibodies requires adherence to specific conditions to maintain activity and prevent degradation:

For long-term storage, aliquoting the antibody into single-use volumes is recommended to avoid repeated freeze-thaw cycles. According to preservation guidelines for biotin-conjugated antibodies, they typically remain stable for up to 12 months at -20°C to -70°C as supplied, for 1 month at 2-8°C after reconstitution under sterile conditions, and for 6 months at -20°C to -70°C under sterile conditions after reconstitution .

How can FkpA antibodies be utilized in cell-free protein synthesis systems?

FkpA has been successfully incorporated into cell-free protein synthesis (CFPS) systems, particularly for the manufacturing of antibody-drug conjugates (ADCs) with site-specific conjugation handles. The methodological approach involves:

• Strain development: Creating specialized E. coli strains with integrated FkpA expression cassettes enhances protein folding capacity in cell-free systems. For example, researchers have integrated tandem copies of FkpA genes with a Pc0 promoter and selection markers into bacterial strains for CFPS applications .

• Expression optimization: The integration procedure typically involves:

  • Construction of FkpA integration cassettes by PCR

  • Electroporation into bacterial strains expressing lambda red recombinase

  • Selection on appropriate antibiotic plates

  • Confirmation of integration by colony PCR and DNA sequencing

• Application in ADC manufacturing: The enhanced folding environment provided by FkpA improves the production of antibodies with site-specific conjugation handles, particularly useful for manufacturing biopharmaceuticals with precise drug-to-antibody ratios .

This methodology is particularly valuable for creating homogeneous antibody conjugates with clearly defined locations for bioconjugation, avoiding the heterogeneity often seen with conventional conjugation methods that rely on natural amino acid functional groups .

What experimental controls are essential when using biotin-conjugated FkpA antibodies?

To ensure reliable and reproducible results with biotin-conjugated FkpA antibodies, the following controls are critical:

For surface protein detection studies, it's particularly important to include controls that distinguish between surface-accessible and periplasmic proteins. One approach involves comparing biotinylated versus non-biotinylated samples using neutravidin pulldown assays followed by tryptic digestion and LC-MS/MS analysis to identify biotin-specific binding proteins .

How can researchers optimize immunoprecipitation protocols using biotin-conjugated FkpA antibodies?

Optimizing immunoprecipitation (IP) with biotin-conjugated FkpA antibodies requires attention to several methodological aspects:

• Lysis buffer optimization:

  • Use buffers containing 0.01M PBS (pH 7.4) supplemented with protease inhibitors

  • Include mild detergents (0.1-0.3% NP-40 or Triton X-100) to solubilize membranes while preserving protein-protein interactions

• Binding conditions:

  • Pre-clear lysates with protein G agarose to reduce non-specific binding

  • Incubate biotin-conjugated FkpA antibodies with lysates for 4-16 hours at 4°C with gentle rotation

  • Use antibody concentrations of 1-5 μg/mL for optimal target capture

• Capture strategies:

  • Use streptavidin-coated magnetic beads rather than agarose for higher purity and less background

  • Implement a two-step binding process: antibody-lysate incubation followed by streptavidin bead capture

  • Wash extensively with decreasing salt concentrations to maintain specific interactions

• Elution methods:

  • For protein complex analysis: Use mild elution with competitive biotin

  • For subsequent immunoblotting: Direct denaturation in SDS sample buffer at 65°C (for detection of SecY) or boiling for 5 minutes (for most other proteins)

• Detection optimization:

  • For Western blot detection, use specific primary antibodies followed by HRP-conjugated secondary antibodies for ECL detection, or alkaline phosphatase conjugates for colorimetric detection with nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate

This optimized approach has been successfully used to isolate and identify FkpA and its interaction partners in bacterial cell lysates with minimal background and high specificity .

How should researchers interpret inconsistent results when using FkpA antibodies across different bacterial strains?

Inconsistent results across bacterial strains may stem from several factors that require systematic analysis:

• Epitope variation:

  • Sequence variations in FkpA across bacterial species or strains may affect antibody recognition

  • Perform sequence alignment analysis of the FkpA protein region containing the immunogen (typically amino acids 26-270 for commercial antibodies)

• Expression level differences:

  • FkpA expression is influenced by growth conditions and stress responses

  • Normalize loading by total protein content and use housekeeping proteins as loading controls

  • Consider qPCR analysis of fkpA gene expression to correlate with protein levels

• Subcellular localization variability:

  • FkpA is primarily periplasmic but its accessibility can vary with membrane permeability

  • Perform fractionation studies to separate periplasmic, membrane, and cytoplasmic fractions

  • Use known compartment markers (e.g., SecY for inner membrane, OmpA for outer membrane)

• Post-translational modifications:

  • Changes in FkpA folding state may affect epitope accessibility

  • Compare results under different sample preparation conditions (native vs. denaturing)

  • Consider chemical crosslinking studies to capture transient conformations

• Systematic analysis approach:

  • Create a matrix of experimental conditions vs. bacterial strains

  • Identify patterns related to Gram-positive vs. Gram-negative, growth phase, or stress conditions

  • Consider FkpA's dual functionality (PPIase and chaperone) when interpreting strain-specific effects

Research by Arié et al. demonstrated that FkpA expression and localization can vary significantly under different stress conditions, which must be considered when comparing results across bacterial strains .

What methodological approaches can distinguish between specific and non-specific binding in complex samples?

Distinguishing specific from non-specific binding requires multiple complementary approaches:

• Competitive inhibition assays:

  • Pre-incubate antibody with purified recombinant FkpA protein before applying to samples

  • Titrate increasing concentrations of competing antigen to demonstrate dose-dependent signal reduction

  • True specific binding shows proportional signal decrease with competitor addition

• Genetic validation:

  • Compare signal in wild-type vs. fkpA deletion mutants

  • Complement deletion strains with plasmid-expressed FkpA to restore signal

  • Use CRISPR-Cas9 knockdown approaches in organisms where gene deletion is challenging

• Epitope mapping:

  • Test antibody against truncated versions of FkpA (N-terminal domain only vs. C-terminal domain only)

  • This approach can also reveal which functional domain of FkpA is recognized

  • Research by Saul et al. showed that deletion mutant FkpNL (N-terminal domain) exists as a mixture of monomeric and dimeric species, while the C-terminal domain is monomeric

• Cross-adsorption controls:

  • Pre-adsorb antibody against lysates from fkpA knockout strains to remove non-specific antibodies

  • Compare signals before and after adsorption to identify truly specific signals

• Orthogonal detection methods:

  • Confirm findings using multiple detection techniques (immunoblotting, mass spectrometry, immunofluorescence)

  • For surface protein detection, compare results from biotinylation-based approaches with other membrane protein isolation techniques

A robust approach used by Myers-Morales et al. involved surface biotinylation followed by neutravidin pulldown with parallel analysis of biotinylated and non-biotinylated samples by LC-MS/MS, allowing clear discrimination between specific and non-specific binding proteins .

What factors affect quantification accuracy when using biotin-conjugated FkpA antibodies?

Accurate quantification with biotin-conjugated FkpA antibodies depends on several critical factors:

• Biotin-to-antibody ratio determination:

  • The number of biotin molecules per antibody affects sensitivity and can lead to steric hindrance

  • Calculate using the formula: Biotin:Antibody ratio = (A350 × 0.9 × MW antibody) ÷ (ε × antibody concentration)

  • Optimal biotin density is typically 2-4 molecules per antibody; higher ratios may cause aggregation or reduced antigen binding

• Antibody concentration measurement:

  • Correct for biotin's contribution to A280 using: Corrected A280 = Measured A280 - (A350 × 0.4475)

  • Calculate antibody concentration: Concentration (μM) = Corrected A280 × 10^6 ÷ 210,000

• Standard curve considerations:

  • Use purified recombinant FkpA at known concentrations as reference standards

  • Include matrix-matched standards that account for sample composition effects

  • Confirm linearity across the expected concentration range with r^2 > 0.98

• Signal detection optimization:

  • Select detection systems appropriate for the expected concentration range

  • For low abundance targets, use amplification systems like tyramide signal amplification

  • For high abundance targets, choose detection methods with broader dynamic range

• Epitope accessibility variables:

  • FkpA conformation affects epitope exposure - the protein shows flexibility in the relative orientation of its C-terminal domains

  • Sample preparation methods influence epitope accessibility and quantification accuracy

  • Consider native vs. denaturing conditions based on the epitope location

Research by Ramm et al. demonstrated that the biotin quantification embedded in modern conjugation kits provides more consistent results than traditional HABA assays for determining biotin incorporation levels, noting that "The HABA assay by comparison is very inconsistent and thus it is much harder to characterize and replicate conjugates" .

How can FkpA antibodies contribute to bacterial pathogenesis research?

FkpA antibodies provide valuable tools for investigating bacterial pathogenesis through several methodological approaches:

• Virulence factor processing studies:

  • FkpA functions as a periplasmic chaperone essential for certain imported toxins

  • For example, research by Stéphanie et al. demonstrated that FkpA is specifically required for the activity of imported colicin M toxin in E. coli

  • The chaperone assists in proper folding of colicin M in the periplasm after it unfolds during import across the outer membrane

• Stress response investigation:

  • FkpA expression changes under various stress conditions relevant to pathogenesis

  • Biotin-conjugated antibodies allow for quantitative tracking of FkpA levels during host-pathogen interactions

  • Deletion of fkpA causes reduced biomass yield (50% reduction at 37°C compared to 10% at 30°C), indicating its importance in temperature stress responses relevant to infection

• Surface protein identification:

  • Surface-exposed proteins represent important vaccine candidates and therapeutic targets

  • Myers-Morales et al. developed a surface biotinylation method using FkpA as a control to distinguish between true surface proteins and periplasmic contaminants in Yersinia pestis

  • This approach identified potential protective antigens for bubonic plague

• Secretion system analysis:

  • FkpA influences the folding of proteins destined for secretion

  • Antibodies against FkpA can reveal secretion bottlenecks in bacterial pathogens

  • Comparative studies between wild-type and fkpA mutants can identify pathogenicity factors dependent on this chaperone

The specificity of biotin-conjugated FkpA antibodies makes them particularly valuable for studying complex host-pathogen interfaces where distinguishing bacterial proteins from host proteins is critical .

What role does FkpA play in antibody-drug conjugate (ADC) manufacturing research?

FkpA has emerged as a key factor in improving cell-free protein synthesis systems for ADC manufacturing:

• Enhancement of folding capacity in CFPS systems:

  • FkpA integration into specialized E. coli strains improves protein folding during cell-free synthesis

  • Researchers have developed strains (e.g., SBDG150) with integrated tandem FkpA genes under constitutive promoters specifically for this purpose

  • The integration method involves PCR-based construction of FkpA cassettes, lambda red recombinase-mediated integration, and selection with appropriate antibiotics

• Site-specific bioconjugation advantages:

  • Traditional ADC production methods result in heterogeneous conjugation sites and drug-to-antibody ratios

  • FkpA-enhanced CFPS systems facilitate the production of antibodies with site-specific conjugation handles

  • This approach enables precise attachment of cytotoxins, fluorophores, radioisotopes, or other functional molecules at defined positions

• Methodological advantages for homogeneous ADCs:

  • FkpA contributes to proper folding of antibodies containing non-natural amino acids (nnAAs)

  • nnAA mutagenesis with amber suppressor technology allows site-specific incorporation of conjugation handles

  • Open nature of CFPS allows direct addition of components of the nnAA incorporation machinery

• Quantification and characterization benefits:

  • Biotin-conjugated antibodies against FkpA can be used to monitor its expression in production strains

  • Spectrophotometric determination of biotin ligand density enables reliable characterization of conjugates

  • This allows researchers to establish optimal biotinylation levels and reproduce successful batches

The integration of FkpA into CFPS systems represents a significant advancement in ADC manufacturing technology, addressing the pharmaceutical industry's "growing desire and market for antibodies with bioconjugation handles at clearly defined locations" .

How does FkpA's dual functionality influence experimental design for protein folding studies?

The dual functionality of FkpA as both a chaperone and peptidyl-prolyl isomerase (PPIase) requires careful experimental design when studying protein folding mechanisms:

• Domain-specific activity separation:

  • The N-terminal domain provides chaperone activity while the C-terminal domain exhibits PPIase activity

  • Deletion mutant studies by Saul et al. demonstrated that FkpNL (N-terminal domain only) maintains chaperone function but lacks PPIase activity

  • Conversely, the C-terminal domain alone shows PPIase activity but no chaperone function

• Inhibitor-based functional discrimination:

  • FK506 specifically inhibits the PPIase activity of FkpA without affecting its chaperone function

  • This allows researchers to distinguish between effects due to each activity

  • For example, FK506 prevents FkpA-mediated renaturation of colicin M but also inhibits FkpA's ability to delay citrate synthase aggregation

• Temperature-dependent experimental designs:

  • FkpA's importance increases at higher temperatures (e.g., 37°C vs. 30°C)

  • Comparative studies at different temperatures can reveal FkpA-dependent folding pathways

  • Research in C. glutamicum showed FkpA deletion causes 50% reduced biomass at 37°C but only 10% reduction at 30°C

• Methodological approaches for studying FkpA-substrate interactions:

  • In vitro folding assays with purified FkpA and model substrates

  • Comparison of wild-type FkpA with point mutations affecting either chaperone or PPIase function

  • Co-precipitation studies to identify substrate preferences for each function

• Structural considerations in experimental design:

  • The V-shaped dimeric structure of FkpA shows flexibility in the relative orientation of C-terminal domains

  • This mobility may adapt FkpA's independent folding functions to different polypeptide substrates

  • Crystal structures in different forms (native, truncated, FK506-bound) provide structural insights for experimental design

Understanding this dual functionality is crucial when interpreting results from FkpA studies, as each function may predominate under different experimental conditions or with different substrate proteins .

What are the potential applications of FkpA antibodies in synthetic biology research?

FkpA antibodies are finding novel applications in synthetic biology through several innovative approaches:

• Designer cell-free protein synthesis systems:

  • Integration of FkpA into specialized E. coli strains enhances folding capacity for CFPS

  • Biotin-conjugated FkpA antibodies enable monitoring and optimization of these systems

  • This approach facilitates production of proteins with site-specific conjugation handles for precise bioconjugation strategies

• Biosensor development:

  • FkpA's substrate binding capability can be exploited for detecting misfolded proteins

  • Antibodies recognizing FkpA-substrate complexes serve as indicators of protein folding stress

  • This system provides a readable output for synthetic circuits responding to protein misfolding

• Protein folding quality control circuits:

  • Synthetic systems incorporating FkpA can improve folding of difficult-to-express proteins

  • Quantitative detection of FkpA expression using biotin-conjugated antibodies allows fine-tuning of these systems

  • The dual functionality of FkpA (chaperone and PPIase activities) provides multifaceted support for protein folding

• Periplasmic pathway engineering:

  • FkpA enhances folding and activity of proteins in the periplasmic space

  • Biotinylated antibodies enable tracking of engineering efforts targeting periplasmic processes

  • Applications include improvement of secretory pathways for recombinant protein production

• Orthogonal protein folding systems:

  • FkpA can be adapted to fold specific target proteins without interfering with host proteostasis

  • Biotin-conjugated antibodies provide tools for detecting and quantifying these orthogonal systems

  • This approach enables the creation of modular, orthogonal folding pathways for synthetic biology applications

The combination of FkpA's well-characterized structure-function relationship and the availability of specific biotin-conjugated antibodies makes it an attractive component for designing robust synthetic biological systems with enhanced protein folding capabilities .

How can researchers integrate FkpA antibody detection with other analytical techniques?

Integration of biotin-conjugated FkpA antibody detection with complementary analytical techniques creates powerful multi-dimensional research platforms:

• Mass spectrometry integration:

  • Immunoprecipitation with biotin-conjugated FkpA antibodies followed by on-bead tryptic digestion

  • Analysis by LC-MS/MS to identify FkpA-interacting proteins

  • This approach was successfully used by Myers-Morales et al. to identify surface-exposed proteins in Y. pestis

• Cryo-electron microscopy coupling:

  • Biotin-conjugated FkpA antibodies combined with gold-labeled streptavidin for localization studies

  • Visualization of FkpA in relation to membrane structures and protein complexes

  • Enhanced resolution of FkpA's dimeric V-shaped structure in its native environment

• Single-molecule techniques:

  • FRET-based approaches using biotin-streptavidin tethering of FkpA to surfaces

  • Observation of real-time conformational changes during substrate binding and catalysis

  • Detection of the flexibility in relative orientation of FkpA's C-terminal domains as observed in crystallographic studies

• Microfluidic systems:

  • Surface immobilization of biotin-conjugated FkpA antibodies in microfluidic channels

  • Real-time monitoring of FkpA expression under different stress conditions

  • High-throughput screening of factors affecting FkpA function and expression

• Bioinformatic integration:

  • Correlation of antibody-based quantification data with transcriptomic profiles

  • Comparative transcriptome analysis in wild-type versus ΔfkpA strains revealed 69 genes with ≥2-fold mRNA level changes in C. glutamicum ΔfkpA

  • This provides insight into the transcriptional response upon mild heat stress when FkpA is absent

The methodological workflow for integrating antibody detection with MS analysis typically involves immunoprecipitation, on-bead digestion, LC-MS/MS analysis, and database searching against both species-specific and comprehensive databases, with careful distinction between specific and non-specific binding proteins .

What technological advances are improving the specificity and sensitivity of FkpA detection?

Recent technological advances have significantly enhanced the specificity and sensitivity of FkpA detection systems:

• Advanced biotin conjugation chemistry:

  • Modern biotin conjugation kits incorporate built-in quantification mechanisms

  • Measurement of absorbance at A350 and A280 allows precise determination of biotin:antibody ratios

  • This enables optimization of conjugation for maximum sensitivity without compromising specificity

• Signal amplification strategies:

  • Polymerized streptavidin detection systems magnify signal from low-abundance targets

  • Biotin-tyramide signal amplification for immunohistochemical applications

  • These approaches maintain specificity while dramatically improving detection limits

• Single-domain antibody fragments:

  • Development of smaller antibody formats targeting FkpA epitopes

  • Enhanced penetration into bacterial periplasmic space

  • Better access to conformational epitopes in the V-shaped dimeric structure of FkpA

• Orthogonal confirmation methods:

  • Combined use of antibodies targeting different epitopes on FkpA

  • Multi-color imaging to distinguish domain-specific interactions

  • Integration with genetic reporters to provide independent verification

• Computational epitope mapping:

  • Structural analysis of FkpA crystal structures to identify optimal epitope targets

  • Design of antibodies targeting conserved regions for cross-species applications

  • Identification of accessible epitopes in both N-terminal (chaperone) and C-terminal (PPIase) domains

• Surface-focused detection strategies:

  • Novel surface biotinylation methods distinguish periplasmic FkpA from surface-exposed proteins

  • Systematic comparison of biotinylated versus non-biotinylated samples to identify truly surface-exposed proteins

  • This approach provides critically important controls for specificity validation in complex bacterial systems

These technological advances collectively support more robust, sensitive, and specific detection of FkpA and its interactions, enabling researchers to address increasingly sophisticated questions about its function in bacterial physiology and pathogenesis .