BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3291

Peptides:

Sequences	Affinity Values
TWPKHFDKHTFYSILKLGKH	[45]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 29090274

Target Name:

Domain 3332-3779 of prolow-density lipoprotein receptor-related protein 1

Template Name:

Alpha-2-macroglobulin receptor-associated protein, Alpha-2-MRAP

Structure of Target-Template Complex:

2FYL

Structure of Target-Peptide Complex:

Not determined.

Library Name:

T7 X12, X16 and X20 phage display library pool

Affinity Measurement Method:

ELISA

Affinity Measurement Description:

Binding activity and selectivity of biotinylated TWPKHFDKHTFYSILKLGKH (L57) to LRP1(3332-3379)-Fc were evaluated by plate ELISA. Binding activity was detected with HRP-conjugated streptavidin and was measured by determining the absorbance at 450 nm. LRP1(3332-3379)-Fc and Act RIIB-Fc are counter proteins. The absorbance at 450 nm of biotinylated L57 was expressed as the mean ± SD (n = 2). The EC50 (nM) value of L57 for binding to LRP1(3332-3379)-Fc was shown.

Brief Description:

For the screening of T7 phage libraries, 20 μg LRP1-Fc was immobilized on Dynabeads Protein A (200 μL; Invitrogen, Carlsbad, CA, USA) in PBS (045–29795; Wako, Osaka, Japan) containing 0.5% BSA. After washing of the beads with PBS containing 0.1% Tween-20 (PBST), they were incubated with 1.0e12 pfu of phage libraries (mixture of X12, X16, and X20) for 1h at room temperature, and then washed with PBST (three times for the first round, five times for the second round, and 10 times for the third to sixth rounds). Bound phages were eluted with 200 μL of 0.5% SDS and then transfected into 80 mL of E. coli BLT5615 cells (Merck Millipore) in the log phase of growth for amplification. After bacteriolysis, the phages were recovered from the culture supernatant by centrifugation and PEG precipitation, according to the T7 Select manual. The recovered phages were dissolved in 1mL of PBS and 0.5 mL of the phages was used for the next round of biopanning.

BiopanningDataSet Comments:

Using phage display technology, we obtained a novel peptide, L57 (TWPKHFDKHTFYSILKLGKH-OH), with an EC50 value of 45 nM for binding to cluster 4 (Ser3332–Asp3779) of LRP1. L57 was stable in mouse plasma for up to 20 min. In situ brain perfusion assay in mice revealed the significantly high BBB permeability of L57

Curator:

Pengcheng Yao

Curation Date:

2018-01-19

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