BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3575

Peptides:

Sequences	Affinity Values
DLFVSSL(18) AETVESC(2) STTGTQY(1) WSLDPSS(1) SLVVERL(1)	[24.3461±1.1215] [NT] [NT] [NT] [NT]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (subtractive panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 33706101

Target Name:

Human embryonic kidney cells 293 (HEK293 cells) transfected with free fatty acid receptor 1 (FFAR1)

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:

Affinity Measurement Description:

HEK293 cells were seeded in a 24-well plate at a density of 1.0e5 cells/well and incubated overnight. Expression vectors (pCAGGS/FFAR1 and pCAGGS/APTGF- a) were kindly provided by Dr. Inoue (Tohoku University) and used for transfection. A mixture of 125 ng pCAGGS/AP-TGF-α and 50 ng pCAGGS/FFAR1 per well in 24-well plates was transfected using PEI (49553-93-7, Polysciences, Warrington, PA, USA) and incubated overnight. The following day, HEK293 cells were harvested by trypsinization and re-seeded in 96-well plates at a volume of 80 mL/well. After incubation for 1 h, 20 mL of palmitic acid (PA) and peptide samples in HBSS containing 5 mM HEPES (pH 7.4) were added and incubated for 1 h. Eighty microliters of supernatant containing released AP-TGF-α was transferred into an empty 96- well plate. A substrate for alkaline phosphatase, paranitrophenylphosphate (p-NPP, 34045, Thermo Fisher Scientific) was added to the transferred medium, and absorbance at 405 nm of the plates was measured using a microplate reader before and after 30 min incubation at 37 ◦C. We calculated AP-TGF-α release as a TGF-α shedding response.

Brief Description:

First, 1.0e7 HEK293 cells in 1 mL phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) were incubated with 1.5e11 phages on a shaking machine (120 rpm) for 2 h at 4 °C. This mixture was centrifuged at 1500 rpm for 1 min, and the supernatant containing phages that could not bind HEK293 cells was incubated with other HEK293 cells. This selection (negative selection) was repeated twice to remove phage-binding HEK293 cells. Next, the supernatant was incubated with 5.0e6 HEK293/FFAR1 cells on a shaking machine (120 rpm) for 2 h at 4 °C. The HEK293/FFAR1 cells were washed five times in cold PBS containing 1% BSA and 0.1% Tween-20. HEK293/FFAR1 cells binding phages were eluted with Glycine-HCl (pH 9.1) containing 1 mg/mL BSA on ice for 10 min. The 10 μL phages were titered according to the manufacturer’s instructions, and the amplified phage using E. coli ER2738 was used in the next round of selection.

BiopanningDataSet Comments:

DLFVSSL showed free fatty acid receptor 1 (FFAR1) agonist activity, although it was lower than that of STTGTQY.

Curator:

Bowen Li

Curation Date:

2023-10-13

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