Human PD-1 Protein, ECD (extracellular domain), Fc-fusion, Biotinylated, Recombinant

Description

Programmed death-1 (PD-1), also known as programmed cell death 1 (PDCD1) or CD279, is a single pass type I transmembrane glycoprotein of the Ig superfamily. It is an immunoreceptor in the CD28/CTLA-4 family with an IgV-type extracellular domain showing 21–33% sequence identity with CTLA-4, CD28 and ICOS.  Members of the CD28/CTLA-4 family have been shown to either promote T cell activation (CD28 and ICOS) or inhibit T cell activation (CTLA­4 and PD­1).  The cytoplasmic domain of PD-1 contains two tyrosine residues, a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). ITIM is widely found in immunoinhibitory receptors and the membrane-proximal tyrosine residue may play a central role for the inhibitory function of PD-1. PD-1 negatively regulates antigen receptor signaling by recruiting protein tyrosine phosphatase upon engaging with either of two ligands, PD-L1 or PD-L2. It inhibits the T-cell proliferation and production of related cytokines such as IL-1, IL-4, IL-10 and IFN-γ. PD-1 is involved in lymphocyte clonal selection and peripheral tolerance, and thus contributes to the prevention of autoimmune diseases. As a negative regulator of T- cell effector mechanisms, PD-1 may lead to suppression of anti-tumor immunity. Blockade of PD-1 inhibitory activity with antibodies enhances anti-tumor immunity in vivo.  PD-1 has been proposed as a promising target for cancer immunotherapy.

 

 

Gene Symbol: PD-1; CD279, PDCD1, PD1,  SLEB2, hPD-1, hPD-l, hSLE1

 

NCBI Gene ID: 5133

 

Uniprot Entry: Q15116

 

Construct Details: The recombinant protein is expressed as a 376-amino acid protein consisting of Pro21 - Thr168 region of PD-1 or CD279 (UniProt accession #Q15116) and a C-terminal Fc from human IgG1, which exists as a homodimer under non-reducing conditions.  

 

Source:  Human cells stably expressing PD-1-Fc and growing in chemical-defined media with no animal components or antibiotics

 

Deduced Amino Acid Sequence:

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFR

VTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTSTGT

HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST

YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

M.W.: Calculated molecular mass 42.1 kDa; estimated by SDS-PAGE under reducing condition 60-65 kDa probably due to glycosylation.  

 

Calculated PI: 8.73

 

Calculated Extinction Coefficients (M-1 cm-1, at 280nm):  55,390

 

Purity: >95% judged by SDS-PAGE under reducing condition (see the gel image above)

 

Formulation: Supplied at 0.5 mg/ml in sterile PBSpH7.4 (carrier- and preservative-free). The purified recombinant protein was labeled with Biotin (3-5 Biotin per molecule) using the standard procedure.

 

Endotoxin Level: <0.1 EU per 1 μg of purified recombinant protein determined by the LAL method

 

Biological Activity: Binds to its ligands PD-L1 (SKU#: FCL0781), PD-L2 (SKU#FCL783) and anti-PD-1 monoclonal antibodies (SKU#: MAB1732, MAB0761, MAB0763). Blocks PD-1’s binding to its ligands, PD-L1 and PD-L2 and their signaling activities. Shows cross-species binding to both human and mouse PD-L1 (SKU#FCL1846)and PD-L2 (SKU#FCL1818) in a functional ELISA (see Technical Data 1-4).  Exhibits immunosuppressive activity in vivo.

 

  

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

Restriction: This product is not transferable or re-sellable.  Customer obtain no right to transfer, assign, or sublicense its use rights, or to transfer, resell, package, or otherwise distribute the product, or to use the product for the benefit of any third party or for any commercial purpose.  Customer may only use the product in compliance with applicable local, state and federal laws, regulations and rules.  Customer may not directly or indirectly use the product or allow the transfer, transmission, export or re-export of all or any part of the product in violation of any export control law or regulation of the united states or any other relevant jurisdiction.  Your use of this product constitutes acceptance of the terms of this limited use agreement.  Please refer to our “terms & conditions” for details.  If you are not willing to accept the limitation of this agreement, G&P Biosciences will accept return of the product for a full/partial refund.

 

 

Technical Data 1: Interaction with Human PD-L1 by ELISA

Recombinant human PD-L1-Fc (SKU#FCL0781) was immobilized at 100 ng/well on a 96-well NuncMaxisorp plate for a functional ELISA with serially diluted, biotinylated human PD-1-Fc (PD-1-Fc-Biot, SKU#FCL0763B), biotinylated human CD80-Fc (CD80-Fc-Biot, SKU#FCL0723B), and biotinylated Fc alone (control Fc-Biot, SKU#FCL00029B). Fc protein alone was used as a negative control to demonstrate the specificity of PD-1:PD-L1 and CD80:PD-L1 interactions under the experimental conditions.  The binding curves were fit and apparent K_D was calculated for PD-1: PD-L1 binding to be 365 ± 38 ng/ml with a linear binding range between 100 and 1500 ng/ml, and apparent K_D for CD80:PD-L1 binding to be 831 ± 101 ng/ml with a linear binding range between 250 and 4000 ng/ml.

 

Technical Data 2:  Cross-species Interaction with to Mouse PD-L1 by ELISA

Recombinant mouse PD-L1-Fc (SKU#FCL1846) was immobilized at 100 ng/well on a 96-well NuncMaxisorp plate for a functional ELISA with serially diluted, biotinylated human PD-1-Fc (PD-1-Fc-Biot, SKU#FCL0763B), biotinylated human CD80-Fc (CD80-Fc-Biot, SKU#FCL0723B), and biotinylated Fc alone (control Fc-Biot, SKU#FCL00029B). Fc protein alone was used as a negative control to demonstrate the specificity of PD-1:mPD-L1 and CD80:mPD-L1 interactions under the experimental conditions.  Mouse PD-L1 shows cross-species reactivity to human PD-1 and CD80. The binding curve was fit and apparent K_D was calculated for PD-1: mPD-L1 binding to be 557 ± 63 ng/ml with a linear binding range between 150 and 2000 ng/ml, and apparent K_D for CD80:mPD-L1 binding to be 9486 ± 5500 ng/ml with a linear binding range between 1500 and 75000 ng/ml. 

 

Technical Data 3: Interaction with Human PD-L2 by ELISA

Recombinant human PD-L2-Fc (SKU#FCL0783) was immobilized at 100 ng/well on a 96-well NuncMaxisorp plate for a functional ELISA with serially diluted, biotinylated human PD-1-Fc (PD-1-Fc-Biot, SKU#FCL0763B) and biotinylated Fc alone (control Fc-Biot, SKU#FCL00029B). Biotinylated Fc protein alone was used as a negative control to demonstrate the specificity of PD-1:PD-L2 interaction under the experimental conditions.  The binding curve was fit and apparent K_D was calculated for PD-1: PD-L2 binding to be 220 ± 9 ng/ml with a linear binding range between 50 and 1000 ng/ml.

 

 

Technical Data 4: Cross-species Interaction with Mouse PD-L2 by ELISA

Recombinant mouse PD-L2-Fc (SKU#FCL1818) was immobilized at 100 ng/well on a 96-well NuncMaxisorp plate for a functional ELISA with serially diluted, biotinylated human PD-1-Fc (PD-1-Fc-Biot, SKU#FCL0763B) and biotinylated Fc alone (control Fc-Biot, SKU#FCL00029B). Biotinylated Fc protein alone was used as a negative control to demonstrate the specificity of PD-1:mPD-L2 interaction under the experimental conditions.  Mouse PD-L2 shows cross-species reactivity to human PD-1 with the highest affinity/avidity among all human and murine PD-1 ligands. The binding curves were fit and apparent K_D was calculated for PD-1: mPD-L2 binding to be 126 ± 14 ng/ml with a linear binding range between 40 and 600 ng/ml.

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

 

 

The product is shipped at 4°C. Upon receipt, centrifuge the product briefly before opening the vial. It is recommended to store small aliquots at the temperature below –20°C for long-term storage and the product is stable for at least 3 months. The undiluted protein can be stored at 4°C for no more than 2 weeks.

 

 

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

 

 

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

 

 

1. EMBO J. 11:3887, 1992   

2. Genomics 23:704-706, 1994

3. Genes Immun. 6:430-437, 2005

4. Immunol. Rev. 229:114-25, 2009

 

 

 

 

Product datasheet (pdf) can be downloaded here: FCL0763B-PDS.pdf

 

 

 

Additional supporting documents, including COA and MSDS are available upon request.

 

 

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

Restriction: This product is not transferable or re-sellable.  Customer obtain no right to transfer, assign, or sublicense its use rights, or to transfer, resell, package, or otherwise distribute the product, or to use the product for the benefit of any third party or for any commercial purpose.  Customer may only use the product in compliance with applicable local, state and federal laws, regulations and rules.  Customer may not directly or indirectly use the product or allow the transfer, transmission, export or re-export of all or any part of the product in violation of any export control law or regulation of the united states or any other relevant jurisdiction.  Your use of this product constitutes acceptance of the terms of this limited use agreement.  Please refer to our “terms & conditions” for details.  If you are not willing to accept the limitation of this agreement, G&P Biosciences will accept return of the product for a full/partial refund.