Recombinant Human Protein disulfide-isomerase protein (P4HB), partial (Active)

What is the molecular structure of recombinant Human P4HB?

Recombinant Human P4HB is a 56-60 kDa protein comprising 491 amino acids (mature form spans Asp18-Lys505). The protein contains two thioredoxin (TRX) domains located at amino acids 25-134 and 368-475, plus an endoplasmic reticulum (ER) retention sequence at amino acids 505-508 . When produced recombinantly, the protein is often tagged (e.g., with a C-terminal 10-His tag) to facilitate purification without compromising its catalytic activity . The protein exhibits high structural conservation across species, with human P4HB sharing approximately 94% amino acid identity with mouse P4HB over amino acids 18-505 .

What are the primary biochemical functions of P4HB in cellular contexts?

P4HB exhibits multiple distinct biochemical functions that vary by cellular location:

This multifunctionality makes P4HB a critical protein in cellular proteostasis and extracellular matrix formation .

What expression systems are optimal for producing functional recombinant P4HB?

Different expression systems yield recombinant P4HB with varying characteristics:

How should recombinant P4HB be stored to maintain optimal activity?

Proper storage is crucial for maintaining P4HB activity. The protein should be stored in appropriate buffer conditions, typically containing:

• Tris buffer

• NaCl

• Glycerol as a cryoprotectant

Storage recommendations include:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Store at -20°C to -70°C for up to 6 months from the receipt date

• Once opened, maintain sterile conditions and store at -20°C to -70°C for up to 3 months

For experiments requiring consistent activity measurements, it's advisable to prepare single-use aliquots to prevent activity loss from repeated freeze-thaw cycles.

How can researchers effectively measure the disulfide isomerase activity of P4HB?

Quantifying P4HB's disulfide isomerase activity requires specialized assays that monitor the protein's ability to catalyze disulfide bond formation, breakage, or rearrangement:

• RNase A refolding assay: This classical method measures P4HB's ability to restore activity to reduced and denatured RNase A by facilitating correct disulfide bond formation. The refolded RNase A activity is monitored spectrophotometrically using RNA or synthetic substrates.

• Di-eosin-glutathione disulfide (di-E-GSSG) assay: A fluorescence-based approach where P4HB-catalyzed reduction of di-E-GSSG results in increased fluorescence as the eosin molecules are separated.

• Insulin turbidity assay: Measures P4HB's reductase activity by monitoring the precipitation of reduced insulin chains, detected as an increase in solution turbidity at 650 nm.

For comparative studies, researchers should standardize their assay conditions, including temperature (typically 25°C or 37°C), pH (optimally 7.0-7.5), and substrate concentrations .

What experimental approaches can distinguish between P4HB's enzymatic and chaperone functions?

Differentiating between P4HB's enzymatic disulfide isomerase activity and its chaperone function requires carefully designed experiments:

• Active site mutations: Generate recombinant P4HB with mutations in the CXXC motifs of the thioredoxin domains to eliminate enzymatic activity while potentially preserving chaperone function.

• Concentration-dependent studies: Exploit the observation that P4HB exhibits chaperone activity at high concentrations but anti-chaperone activity at low concentrations. Design experiments with varied P4HB concentrations and measure protein aggregation using light scattering or centrifugation techniques .

• Phosphorylation studies: Investigate how phosphorylation by FAM20C enhances P4HB's chaperone activity using either phosphomimetic mutations or in vitro phosphorylation systems.

• Chemical inhibition: Utilize specific PDI inhibitors such as PACMA 31, an irreversible inhibitor that preferentially targets the enzymatic function, allowing researchers to isolate the chaperone activity .

How does P4HB contribute to collagen biosynthesis as part of the prolyl 4-hydroxylase complex?

P4HB serves as the beta subunit (also called the PDI subunit) of prolyl 4-hydroxylase (P4H), forming a tetrameric α₂β₂ complex with the alpha subunits. To study this function:

• Reconstitution experiments: Combine purified recombinant P4HB with alpha subunits to form the active P4H tetramer in vitro. Measure hydroxylation activity using synthetic collagen peptides containing proline residues.

• Co-immunoprecipitation studies: Use antibodies against P4HB to pull down the entire P4H complex from cellular lysates, followed by mass spectrometry to identify interacting partners and post-translational modifications.

• Oxygen consumption assays: Since P4H is an oxygen-dependent enzyme, measure oxygen consumption rates as an indicator of hydroxylation activity.

• Ascorbate dependency: P4H requires ascorbate (vitamin C) as a cofactor, and researchers can manipulate ascorbate levels to modulate P4H activity in experimental systems .

What control experiments are essential when studying P4HB in cellular systems?

Robust experimental designs for P4HB studies should incorporate these controls:

• Catalytically inactive mutants: Compare wild-type P4HB with active-site mutants (typically mutations in the CXXC motifs) to distinguish between enzymatic and structural roles.

• Dose-response experiments: Include multiple concentrations of P4HB to account for its concentration-dependent switch between chaperone and anti-chaperone activities .

• Subcellular localization controls: Use compartment-specific markers alongside P4HB to distinguish between its ER and cell surface functions.

• Species-specific considerations: When extrapolating between models, account for the 6% sequence difference between human and mouse P4HB (amino acids 18-505) .

• Post-translational modification status: Verify the phosphorylation state of P4HB, particularly when studying its chaperone function, which is enhanced by FAM20C-mediated phosphorylation .

How can researchers effectively study P4HB's role at the cell surface versus its intracellular functions?

P4HB performs distinct functions in different cellular locations. To study location-specific functions:

• Cell surface biotinylation: Use membrane-impermeable biotinylation reagents to selectively label and isolate cell surface P4HB for functional studies.

• ER retention sequence mutations: Generate P4HB constructs with mutations in the ER retention sequence (amino acids 505-508) to increase cell surface localization .

• Live-cell imaging: Employ fluorescently tagged P4HB constructs with appropriate controls to visualize its trafficking between compartments.

• Receptor interaction studies: Investigate P4HB's interaction with LGALS9 at the cell surface of Th2 T helper cells, which retains P4HB at the plasma membrane and enhances cell migration through altered redox states .

How does P4HB contribute to viral infection mechanisms?

P4HB's cell surface reductase activity has implications for viral entry, particularly for HIV:

• Viral entry assays: Measure viral entry efficiency in the presence of P4HB inhibitors or after P4HB knockdown/knockout.

• Thiol-disulfide exchange monitoring: Employ redox-sensitive fluorescent probes to visualize P4HB-mediated reduction of viral disulfide bonds.

• Cell-based fusion assays: Quantify the ability of P4HB to facilitate HIV fusion with CXCR4-expressing cells through its reduction of viral disulfide bonds .

This research area has potential therapeutic implications, as targeting P4HB might represent a novel approach to preventing viral entry.

What experimental designs are appropriate for studying the role of P4HB in disease models?

P4HB has been implicated in various pathological conditions, including Cole-Carpenter Syndrome. Researchers can employ:

• Patient-derived cells: Compare P4HB expression, localization, and activity in cells from affected individuals versus controls.

• CRISPR/Cas9 gene editing: Introduce disease-associated mutations into cellular models to study their effects on P4HB function.

• Animal models: Develop conditional knockout or knockin mouse models to study tissue-specific effects of P4HB alterations.

• Proteostasis challenges: Expose cells with altered P4HB function to ER stress inducers to assess changes in the unfolded protein response pathways.

• High-throughput compound screening: Identify modulators of P4HB activity that could potentially correct disease-associated defects in protein folding .

What statistical approaches are recommended for analyzing P4HB activity data?

When analyzing P4HB activity data, researchers should consider:

• Enzyme kinetics modeling: Apply Michaelis-Menten or more complex enzyme kinetics models to determine parameters such as Km and Vmax for different substrates.

• Concentration-dependent effects analysis: Use non-linear regression to analyze the biphasic effects of P4HB concentration on protein aggregation.

• Comparative activity analysis: Employ ANOVA with appropriate post-hoc tests when comparing wild-type versus mutant P4HB activity or P4HB from different sources.

• Time-course experiments: Apply repeated measures analysis for time-dependent changes in P4HB activity.

• Active learning approaches: For complex experimental designs with multiple variables, consider advanced machine learning techniques for experimental design optimization as described in computational biology research3.

How can researchers optimize experimental design when studying complex P4HB interactions?

When investigating P4HB's multiple functions and interactions:

This methodological approach ensures efficient use of resources while maximizing the information gained from each experiment, particularly important when working with recombinant proteins that may be costly or challenging to produce in large quantities.