Identification Of A CD36 Scavenger Receptor Binding Peptide Using A 12-MER Peptide M13 Phage Display Library
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Date
2018-01
Authors
Ayob, Muhammad Salahuddin
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Publisher
Universiti Sains Malaysia
Abstract
CD36 is a class B scavenger receptor which is involved significantly in the uptake of oxidised low-density lipoprotein (oxLDL). Accumulation of lipid contributes to foam cells formation, which is the precursor to atherosclerosis plaque. The aim of this study was to identify specific peptides which interact and bind to CD36 as it has the potential to be developed into a peptide inhibitor and as a molecular intervention in treating athero-inflammatory disorders. In this study, 12-mer Ph.D-12TM Phage Display Peptide Library, which was validated in streptavidin panning, was used in selection against CD36. CD36-binding assay was performed by incubating the selected phages with two CD36-expressing cell lines, CHO-CD36 and U937 and a CD36-non expressing cell line, HeLa, followed by anti-M13 phage-PE and mouse IgG2b-PE isotype antibodies staining. All stained cells were acquired on BD FACS CaliburTM flow cytometer and analysed using FlowJo 7.6.5 software. Binding specificity was assayed using cell-based ELISA. The phage clones were incubated with the cells and stained with anti-M13-HRP. TMB was used in phage detection and the signal was read using a microplate reader. Secondary structure of CD36 phage and binding interaction of the peptide to CD36 receptor were predicted using I-TASSER and CABS-dock software, respectively. Phage bearing specific streptavidin motif, HPM, was selected from streptavidin panning and validated the library used in the selection. Fourth round of CD36 panning yielded unique phage clones that had no common motif with any published peptide when analysed using online database Target Unrelated Peptide Scanner (SAROTUP) and Mimotope Database and Beyond (MimoDB).
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Keywords
A CD36 scavenger receptor binding peptide , using a12-MER peptide M13 phage display library