Peptidyl-prolyl cis-trans isomerase A/CYPA Protein, Human (Tag Free)

CYPA protein, a peptidyl prolyl cis-trans isomerase, has proinflammatory activity by stimulating activation of NF-kappa-B and ERK, JNK, and p38 MAP kinases. It may act as a mediator between the human SARS coronavirus nucleoprotein and BSG/CD147 during the virus' invasion of host cells. Peptidyl-prolyl cis-trans isomerase A/CYPA Protein, Human is the recombinant human-derived Peptidyl-prolyl cis-trans isomerase A/CYPA protein, expressed by E. coli , with tag free.

Peptidyl-prolyl cis-trans isomerase A (CYP A) catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides. This enzyme plays a multifaceted role, exerting a strong chemotactic effect on leukocytes through the activation of its membrane receptor BSG/CD147, initiating a signaling cascade culminating in MAPK/ERK activation. Additionally, CYP A activates endothelial cells (ECs) in a pro-inflammatory manner, inducing NF-kappa-B and MAP-kinase signaling, and upregulating adhesion molecules. It induces apoptosis in ECs, promoting FOXO1-dependent expression of CCL2 and BCL2L11. In response to oxidative stress, CYP A initiates proapoptotic and antiapoptotic signaling in ECs. It negatively regulates MAP3K5/ASK1 kinase activity and is necessary for the assembly of TARDBP in heterogeneous nuclear ribonucleoprotein complexes, regulating TARDBP binding to RNA. CYP A also plays a crucial role in platelet activation and aggregation, regulates calcium mobilization, and facilitates integrin ITGA2B:ITGB3 bidirectional signaling. Moreover, it inhibits replication of influenza A virus and may act as a mediator between the SARS coronavirus nucleoprotein and BSG/CD147 during virus invasion of host cells.

Measured by its ability to inhibit calcineurin phosphatase activity in the presence of Cyclosporin A. The IC₅₀ for inhibition of calcineurin activity is ≤420 nM that incubate at 37 ºC for 60 minutes.

Materials
Assay buffer: 20 mM Tris, 10 mM MgCl₂, 0.1 mM CaCl₂, 1 mg/mL BSA, pH 7.5
Test protein: Peptidyl-prolyl cis-trans isomerase A/CYPA Protein, Human (Tag Free) (HY-P70047A)
Calcineurin Protein, Human
Calmodulin Protein, Human (HY-P7710)
Substrate: Serine/Threonine Phosphatase Substrate I, 10 mM stock solution in deionized water
Cyclosporin A, 1 mM stock solution in DMSO
Malachite Green Phosphate Assay Kit

Procedure
1. Dilute Calcineurin to 1.17 μg/mL, Calmodulin to 16.8 μg/mL, and Cyclosporin A to 30 μM in the assay buffer.
2. Dilute Human CYPA to the following concentrations: 20000, 10000, 5000, 2500, 1250, 625, 313, 156, 78, 39, and 20 nM.
3. Add the following to the microplate wells:
Sample group: 5 μL of Human CYPA dilution, 5 μL Calmodulin, 5 μL of Cyclosporin A, 25 μL of Calcineurin.
Control group: 5 μL of assay buffer, 5 μL Calmodulin, 5 μL of Cyclosporin A, 25 μL of Calcineurin.
4. Seal the plate, mix by tapping, and incubate at room temperature for 1 hour.
5. After incubation, remove the seal and add 10 μL of 1 mM substrate. The total volume should be 50 μL.
6. Re-seal the plate and incubate with shaking at 37°C for 1 hour. Adjust the volume to 200 μL with assay buffer.
7. Add 70 μL of chromogenic reagent to each well, gently mix by pipetting up and down, and allow to stand at room temperature for 30 minutes.
8. Measure at 630 nm (absorbance) in endpoint mode.
9. Determine the 50% inhibitory concentration (IC₅₀).

Human

E. coli

Tag Free

P62937-1 (M1-E165)

5478

Full Length

MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE

Approximately 18 kDa, based on SDS-PAGE under reducing conditions.

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.

• [1]. /biology-dictionary/a549-cell.html  [Content Brief]