BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3607

Peptides:

Sequences
WSLGYTG(4/20) GTIYWNS(1/20) SPLSPRY(1/20) ETGITRQ(1/20) WIFTPLG(1/20) RNSWPVW(1/20) GSSGKPG(1/20) RGTGHYW(1/20) WWSTHDR(1/20) RNMRGYG(1/20) WTARPTG(1/20) GSWTTGQ(1/20)

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 33481966

Target Name:

T-cell surface glycoprotein CD3 zeta chain

Template Name:

Not determined.

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

Ph.D.-7 phage display library (X7)

Affinity Measurement Method:

Flow Cytometry

Affinity Measurement Description:

All candidate phage clones harvested from the biopanning experiments and two random clones from the stock library were amplified, tittered, and plated for the determination of each concentration (PFU/ml). Streptavidin-coated iron-oxide microbeads (Dynabeads M280, Invitrogen) were decorated with biotinylated human CD3e (Avitag) at a saturation density (>10 μg of biotinylated protein per 1 mg of Dynabeads). CD3e-coated beads were washed with PBS buffer once and incubated with candidate phage clones (10^12 virions per 1 mg beads) in 200 μl TBST buffer (tris-buffered saline with 0.1% tween) for 30 minutes at room temperature on a shaker. After washing the beads with FACS buffer once, the beads were incubated with anti-M13 Major Coat Protein antibody (1 : 100 dilution) for 30 minutes at room temperature on a shaker. The bead samples were finally washed and resuspended in FACS buffer to be examined using flow cytometry.

Brief Description:

Screening of the PhD-7 phage display peptide library on human recombinant CD3ε protein as the target protein was carried out according to the manufacturer's protocol with some modifications. Briefly, 50 μl of a 50% aqueous suspension of Pierce protein A/G magnetic beads was washed with 1 ml of TBST washing buffer (tris-buffered saline with 0.1% tween) on a magnetic separator and blocked in blocking buffer (0.1 M NaHCO3 (pH 8.6) with 0.5% (w/v) BSA) for 1 h at 4 °C on a rotator. 2 picomoles of target protein was mixed with 1011 pfu (plaque-forming unit) of the phage library in 200 μl of TBST buffer and incubated for 15 minutes at room temperature on a rotator and then incubated with pre-washed and pre-blocked magnetic beads for 15 minutes at room temperature on a rotator. After the microbeads were washed vigorously 15 times with 1 ml TBST, the bound phage was eluted by 1 ml of glycine buffer (0.2 M glycine–HCl (pH 2.2) + 0.1% (w/v) BSA) and neutralized by the addition of 150 μl of 1 M Tris–HCl (pH 9.1). The harvested phages were amplified by infecting E. coli (ER2738) cells and precipitated using PEG8000/NaCl solution. After each round, a negative selection was performed by incubating the harvested phage clones from the previous round with the empty protein A/G magnetic beads in TBST buffer for 20 minutes at room temperature on a rotator. Only the unbound phages in this negative selection were used for the next rounds of panning. After the third and the fifth round of biopanning, the resulting phage clones tittered on LB/IPTG/Xgal plates, and 20 out of 100 blue plaques were randomly selected for sequencing.

BiopanningDataSet Comments:

WSLGYTG demonstrated a superior binding behavior to other clones in the binding assays against recombinant T-cell surface glycoprotein CD3 zeta chain (CD3 zeta) on microbeads or Jurkat cells. The synthesized peptide also showed specific binding to Jurkat cells in a dose-dependent manner but not to B cell lymphoma line, 2PK3 cells. Molecular modeling and docking simulation confirmed that the selected peptide ligand in an energetically stable conformation binds to a pocket of CD3 zeta that is not hidden by either CD3 gamma (T-cell surface glycoprotein CD3 gamma chain) or CD3 delta (T-cell surface glycoprotein CD3 delta chain). Lastly, magnetic microbeads conjugated with the synthesized peptide ligands showed a weak but specific association with Jurkat cells and induced the calcium flux, a hallmark indication of proximal T cell receptor signaling, which gave rise to an enhancement of IL-2 section and cell proliferation. The novel peptide ligand and its various multivalent forms have a great potential in applications related to T cell biology and T cell immunotherapy.

Curator:

Bowen Li

Curation Date:

2023-10-13

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