Recombinant Human P4HB Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human P4HB Protein, CF Summary

• Details of Functionality: Measured by its ability to promote aggregation of insulin in the presence of DTT. The specific activity is >7.5 A₆₅₀/cm/min/mg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Protein Disulfide Isomerase/P4HB protein
  Asp18-Lys505, with a C-terminal 10-His tag
• Accession #: P07237
• N-terminal Sequence: Asp18
• Protein/Peptide Type: Recombinant Enzymes
• Gene: P4HB
• Purity: >80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 56 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 60 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Buffer A: 100 mM NaH₂PO₄, 3 mM DTT, pH 7.0
  • Buffer B: 100 mM NaH₂PO₄, 3 mM EDTA, pH 7.0
  • Recombinant Human Protein Disulfide Isomerase/P4HB (rhP4HB) (Catalog # 4236-DI)
  • Insulin (Sigma, Catalog # I-5500), 10 mg/mL solution in 50 mM Tris, pH 7.5 (solution will be cloudy)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute insulin to 1.5 mg/mL in Buffer B.
  2. Incubate diluted insulin for 10 minutes at room temperature (insulin will solubilize).
  3. Dilute rhP4HB to 45 µg/mL in Buffer A.
  4. Mix 100 µL of 45 µg/mL rhP4HB and 200 µL of 1.5 mg/mL insulin. Create a Substrate Blank with 100 µL Buffer A and 200 µL insulin.
  5. Incubate at room temperature for 20 minutes.
  6. Load in a clear 96-well plate 100 µL from each reaction vial in duplicate.
  7. Read at 650 nm (absorbance) in kinetic mode for 5 minutes.
  8. Calculate specific activity:

  Specific Activity (Abs/cm/min/mg) = (Adjusted Vmax* (Abs/min)**) / (path corr.(cm)*** x amount of enzyme (mg))

  *Adjusted for Substrate Blank
  **Note: the output of many spectrophotometers in kinetic mode is in mOD.
  ***Using the pathlength correction of 0.32 cm

  Per Well:
  • rhP4HB: 0.0015 mg
  • Insulin: 0.100 mg

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human P4HB Protein, CF

• Cellular thyroid hormone-binding protein
• collagen prolyl 4-hydroxylase beta
• DSI
• ERBA2L
• GIT
• glutathione-insulin transhydrogenase
• P4HB
• P4Hbeta
• p55
• PDI
• PDIA1
• PDIA1procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), betapolypeptide (protein disulfide isomerase-associated 1)
• PDIEC 5.3.4.1
• PHDB
• PO4DB
• PO4HB
• procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), betapolypeptide
• PROHB
• PROHBprocollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), betapolypeptide (protein disulfide isomerase; thyroid hormone binding protein p55)
• Prolyl 4-hydroxylase subunit beta
• prolyl 4-hydroxylase, beta polypeptide
• protein disulfide isomerase family A, member 1
• protein disulfide isomerase/oxidoreductase
• protein disulfide isomerase-associated 1
• protein disulfide-isomerase
• protocollagen hydroxylase
• Thbp
• thyroid hormone-binding protein p55

Background

Protein Disulfide Isomerase, also known as prolyl 4‑hydroxylase subunit beta (P4HB), protocollagen hydroxylase, cellular thyroid hormone binding protein p55 and glutathione-insulin transhydrogenase (1-3) is an abundant multifunctional enzyme that belongs to the Protein Disulfide Isomerase family. It contains two thioredoxin domains that catalyze the formation, breakage and rearrangement of disulfide bonds. When present as a tetramer consisting of two alpha subunits and two beta subunits, this enzyme functions as a hydroxylase catalyzing the hydroxylation of prolyl residues in preprocollagen. P4HB has various additional functions (4-7). It binds thyroid hormone. It acts as a chaperone that inhibits aggregation of misfolded proteins. It plays a role in both the influx and efflux of S-nitrosothiol-bound nitric oxide. It is also a subunit of the microsomal triglyceride transfer protein complex.

1. Pihlajaniemi, T. et al. (1987) EMBO J. 6:643.
2. Cheng, S.Y. et al. (1987) J. Biol. Chem. 262:11221.
3. Morris, J. I. and Varandani, P. T. (1988) Biochim. Biophys. Acta 949:169.
4. Obata, T. et al. (1988) J. Biol. Chem. 263:782.
5. Gilbert, H. F. (1997) J. Biol. Chem. 272:29399.
6. Sliskovic, I. et al. (2005) J. Biol. Chem. 280:8733.
7. Wetterau, J. R. et al. (1990) J. Biol. Chem. 265:9800.

Publications for Protein Disulfide Isomerase/P4HB (4236-DI)(1)

Publication using 4236-DI

• Huang, R;Wu, D;Zhang, K;Hu, G;Liu, Y;Jiang, Y;Wang, C;Zheng, Y; ARID1A loss induces P4HB to activate fibroblasts to support lung cancer cell growth, invasion, and chemoresistance Cancer science 2023-12-15 [PMID: 38100120] (Bioassay, Human)

Bioinformatics

• Gene Symbol: P4HB
• Uniprot:
  • P07237()