BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3596

Peptides:

Sequences	Affinity Values
AAHRVGGFNYHM(7/20) SVPLNSWSIFPR(5/20) STVYHTTPYHNR(3/20)	[1097.7413±82.1182] [1526.2958±108.2934] [NT]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 37098917

Target Name:

Staphylococcus aureus, S.aureus

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:

ELISA

Affinity Measurement Description:

An ELISA plate was coated with an S. aureus suspension using the methods described in “Two panning methods for phage selection,” above. Serial dilutions of the phage in 100 μL of TBST were prepared, added to each well, and then incubated at RT for 1 h with agitation. The plate was washed six times with TBST; then, 200 μL of the diluted HRP-conjugated anti-M13 monoclonal antibody (GE Healthcare) was added to each well and incubated at RT for 1 h with agitation. The HRP substrate solution was prepared according to the manufacturer’s instructions. Finally, 200 μL of substrate solution was added to each well and incubated for 60 min at room temperature (RT) with gentle agitation, and the plate was read at 405 to 415 nm.

Brief Description:

Staphylococcus aureus cells were cultured in Luria-Bertani (LB) medium until the optical density at 600 nm (OD600) reached 0.6 to 0.8. The cells were then collected by centrifugation, washed with phosphate-buffered saline (PBS), and mixed with ethanol before being dried on a 96-well plate (100 μL/well) at room temperature (RT) overnight. For the first panning round, the cells fixed on the 96-well plate were exposed to e11 PFU/mL of phage library in TBST buffer (Tris-buffered saline with 0.05% Tween 20, pH 7.5) (100 μL/well). Unbound phages were eliminated by washing five times with TBST, and the bound phages were eluted with 100 μL elution buffer (0.2 M Glycine-HCl) for 20 min at room temperature. The eluted phage was neutralized with 1 M Tris-HCl (pH 9.1) and amplified in E. coli ER2738, followed by purification with polyethylene glycol precipitation.

BiopanningDataSet Comments:

A phage clone displaying a peptide capable of specific binding to a whole S. aureus cell was selected from a 12-mer phage peptide library. The peptide sequence was SVPLNSWSIFPR. The selected phage’s ability to bind specifically with S. aureus was confirmed using an enzyme-linked immunosorbent assay, and the chosen peptide was then synthesized. The results showed that the synthesized peptides displayed high affinity with S. aureus but low binding ability with other strains, including Gram-negative and Gram-positive bacteria such as Salmonella sp., Shigella spp., Escherichia coli, and Corynebacterium glutamicum. In addition, yeast vacuoles were used as a drug carrier by encapsulating daptomycin, a lipopeptide antibiotic used to treat Gram-positive bacterial infections. The expression of specific peptides at the encapsulated vacuole membrane created an efficient system that can specifically recognize and kill S. aureus bacteria.

Curator:

Bowen Li

Curation Date:

2023-10-13

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