BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3576

Peptides:

Sequences	Affinity Values
WDMWPSMDWKAE(4/27) FGLEPRANLHFT(2/27) VQVRDNLPTTTG(2/27) THDMGKMDRTTT(2/27) WGNSQWTGQHTT(1/27)	[1.0403±0.0736] [0.6586±0.0164] [0.2498±0.0172] [0.307±0.0265] [0.7087±0.0236]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (subtractive panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

2-3

Reference:

PMID: 35168735

Target Name:

Interleukin-33 (IL-33)

Template Name:

Interleukin-1 receptor-like 1

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:

ELISA

Affinity Measurement Description:

ELISA was performed to evaluate the binding affinity of the phage particles to IL-33. First, streptavidin-coated wells were incubated with 100 μL of biotinylated IL-33 (1 μM) at room temperature for 1 h. The supernatant with residual proteins or unbound phages was subsequently discarded, and then, 200 μL of blocking buffer comprising 0.1 M NaHCO3 (pH 8.6) with 5 mg/mL of BSA and 0.02% NaN3, was added to the wells and incubated for 1 h at 4 °C. The wells were filled with blocking buffer. After incubation, the plate wells were washed 6 times with 0.5% PBST (0.1 M PBS (pH 7.4) with 0.5% Tween 20). The screened peptide-displaying phages (1 × 1011 or 1 × 1012 pfu/mL) were added to the pre-functionalized wells and incubated for 1 h at room temperature with mild shaking. The phage solutions were removed, and then the wells were washed six times with 0.5% PBST to remove unbound phages. The wells were then filled with HRP-conjugated anti-M13 monoclonal antibodies (diluted 1:5000 in blocking buffer) and incubated for 1 h at room temperature with mild shaking. Finally, the wells were washed with 0.5% PBST and filled with 200 μL ABTS (with H2O2), which is an HRP substrate. The change of absorbance was measured at 405 nm using a microplate spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA). The change of absorbance at 405 nm was reproduced from Figure 1A.

Brief Description:

We performed three rounds of biopanning against IL-33 protein. A negative selection beginning with the 2nd round, in which the amplified phage was preincubated with the streptavidin-coated plate in the absence of IL-33, was performed.

BiopanningDataSet Comments:

The selected IL-33 specific peptide was identified as FGLEPRANLHFT. To investigate the molecular interactions between IL-33 and the affinity peptide, the peptide was separated from the phage particles, chemically synthesized and characterized by square wave voltammetry (SWV), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST). The binding constant (Kd) value with SWV, MST, and ITC was found to be 1.68 ± 0.37 μM, 5.98 ± 1.30 μM, and 2.68 ± 1.37 μM, respectively. Two-dimensional (2D) NMR spectral analysis was performed to elucidate the primary peptide binding site of IL-33, which was near the ST2-D3 and IL1RAcP-D3 binding interfaces.

Curator:

Bowen Li

Curation Date:

2023-10-13

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