BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3386

Peptides:

Sequences
SGVYKVAYDWQH(11) GLHTSATNLYLH(2) TVLSHPSTATLI(1) QQRPYVQDLRLI(1)

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (subtractive panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 30387183

Target Name:

BDNF/NT-3 growth factors receptor (EC:2.7.10.1)[1-430]

Template Name:

Brain-derived neurotrophic factor, BDNF

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

Ph.D.-12 phage display library (X12)

Affinity Measurement Method:

ELISA

Affinity Measurement Description:

The phage particles were added to microtiter wells coated with 10 µg/mL of NTRK2, p75NTR (as nonspecific bindings), or bovineserum albumin (BSA; as a negative control) in coating buffer. The plate was washed three times after 1 hour incubation and 50µL of an anti-M13 antibody-horseradish peroxidase conjugate (GE Healthcare) was added. Finally, 50 µL of a substrate chromogen (TMB) was added and the plate was incubated at room temperature in the dark. Absorbance at 450nm was measured using Multiskan ELISA reader. The specificities and affinities of phage-displayed peptides were shown in Figure 3.

Brief Description:

In the first round, the Ph.D‐12 peptide library (New England Biolabs, Herts, UK) was added to 600 ng of recombinant BDNF/NT-3 growth factors receptor in 200 µL of tris‐buffered saline Tween 20 (TBS/T; 0.1% [vol/vol] Tween 20) and incubated for 2 hours. Then, 50 μL of protein G magnetic beads (Protein G Mag Sepharose; GE Healthcare UK Ltd, Little Chalfont, Buckinghamshire, UK) were added. After incubation and magnetic separation, the supernatant was removed (containing unbound phage). The magnetic beads were washed eight times with 1mL of TBS/T (0.1%) to remove weakly‐or nonspecifically‐bound phage. In the second round, one part of the amplified phage stock from the first round was preincubated with the beads in the absence of target to exclude protein G‐binding phage particles. Then, the unbound phage fraction was incubated with recombinant human BDNF/NT-3 growth factors receptor and specifically. The stringency of panning was increased in the third round of panning using reduced concentration of target protein and increased concentration of detergent. Also, a further round of biopanning was carried out and the phage particles were preincubated with recombinant human p75NTR ((nerve growth factor receptor/TNFRSF16 Fc Chimera; R&D System, Minneapolis, MN).

BiopanningDataSet Comments:

The conserved residues of loop2 of brain-derived neurotrophic factor (BNDF) including S32‐GTVLKVPVQ‐L49 shared the most identity with the peptide 1 sequence (SGVYKVAYDWQH). Also, GLAM2SCAN algorithm, RRID:SCR_00178334 introduced peptide 1 as a motif due to the most frequency and similarity with BDNF. In silico molecular docking showed strong interactions between the peptide three dimensional models and the surface residues of BDNF/NT-3 growth factors receptor at the IgC2 domain. A consensus peptide sequence was then synthesized to generate a mimetic construct (named as RNYK). The affinity binding and function of this construct was confirmed by testing against the native structure of BDNF/NT-3 growth factors receptor in SH-SY5Y cells in vitro using flow-cytometry and MTT assays, respectively. RNYK at 5 ng/mL prevented neuronal degeneration of all trans-retinoic acid-treated SH-SY5Y with equal efficacy to or even better than BDNF at 50 ng/mL.

Curator:

Ying Wang

Curation Date:

2019-01-29

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