BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3624

Peptides:

Sequences	Affinity Values
VLGREEWSTSYW LEKGNTLSTSTV NPIVRSAEDGQL NESGITRIALQD YDADMFYMKTNM	[10.6105] [NT] [NT] [NT] [NT]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Immumina iseq100

Sequencing Method:

Next-generation sequencing

Rounds of Panning:

Reference:

PMID: 36456600

Target Name:

Extracellular domain of epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII)

Template Name:

Not determined.

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:

Flow Cytometry

Affinity Measurement Description:

Brief Description:

Ph.D.™-12 Phage Display Peptide Library Kit was purchased from New England Biolabs Inc. (Beverly, MA, USA). Biopanning procedures were done according to the manufacturer’s instruction with certain modifications. Briefly, a 96-well plate was coated with 150 µL hEGFRvIII (Sino Biological #29662-H08B) (in the first two rounds at 100 μg/mL, the third round at 10 μg/mL) overnight at 4 °C. Wells were washed with PBS, blocked with blocking buffer (3% BSA in PBS), washed six times with cold PBST (PBS + 0.1% [v/v] Tween-20), then incubated with 1010 pfu phage peptide library Ph.D.™-12 for 2 h at RT. Unbound phages were removed by washing 10 times with cold PBST (PBS + 0.1% [v/v] Tween-20 in the first round and 0.5% [v/v] in the other two rounds). Bound phages were eluted with 100 μL of 0.2 M Glycine–HCl (pH 2.2), 1 mg/mL BSA and neutralized with 15 μL of 1 M Tris–HCl, pH 9.1. The elution procedure was repeated three times and the final eluate was used for amplification in Escherichia coli ER2738 culture. Recovered phages were subjected for two more rounds of biopanning with hEGFRvIII proteins (Sino Biological #29662-H08B). The eluates of each round were titrated by qPCR to enumerate phages and eluate from the third round of screening for NGS sequencing. Streptavidin was used as a biopanning control with the same condition as the target except for elution. The unbound phages were eluted with 100 μL of 0.1 M Biotin in PBS for 30 min.

BiopanningDataSet Comments:

The enriched peptides were characterized and their binding capacity towards stable cell lines expressing cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII), EGFR wild type (EGFR WT), or a low endogenous level of EGFR WT was confirmed by flow cytometry analysis. The best peptide candidate, VLGREEWSTSYW, was synthesized, and its binding specificity towards EGFRvIII was validated in vitro. Additionally, computational docking analysis suggested that the identified peptide binds selectively to EGFRvIII. The novel VLGREEWSTSYW peptide is thus a promising EGFRvIII-targeting agent for future applications in cancer diagnosis and therapy.

Curator:

Bowen Li

Curation Date:

2023-10-13

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