CD36 binding ability of selected peptide phages identified by using phage display technique
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1
Universiti Sains Malaysia, Regenerative Medicine Cluster, Advanced Medical and Dental Institute (AMDI), Malaysia
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2
Universiti Sains Malaysia, Infectomics Cluster, Advanced Medical and Dental Institute (AMDI), Malaysia
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3
Universiti Sains Malaysia, School of Biological Sciences, Malaysia
CD36 is a class B scavenger receptor and a significant uptake route for oxidised low-density lipoprotein (oxLDL). Accumulation of lipids contributes to foam cells formation, which is the precursor to atherosclerotic plaque. Therefore, CD36-targeting peptides have the potential to be developed as a molecular intervention in treating athero-inflammatory disorders. In this study, Phage display selection Ph.D-12TM Phage Display Peptide Library (NEB) was used in selection against plastic and BSA (negative selection), followed by human CD36 protein (positive selection). All incubation was carried out for 1 hour at 4°C. Bound phages were eluted using oxLDL buffer, amplified and used in subsequent round of panning. After 4th panning, phage clones were selected. The phage DNA were sent for sequencing. CD36 expression CHO-CD36, HeLa and U937 cells were stained with anti-hCD36-FITC and mouse IgG1-FITC isotype antibodies. CD36 binding test Cells were incubated with different phage clones followed by anti-M13 phage-PE and mouse IgG2b-PE isotype antibodies staining. Streptavidin-specific phage was included as an irrelevant phage control. All stained cells were acquired on BD FACSCalibur™ flow cytometer and analysed using FlowJo 7.6.5 software. The results showed that all peptide sequences, which were analysed using online peptide databases Target Unrelated Peptide Scanner (Sarotup) and Mimotope Database and Beyond (MimoDB), are unique and have no common motif with any published peptides nor amongst the clones selected (data not shown). CHO-CD36 and U937 cells showed high level of CD36 expression i.e. 99.7% and 99.4%, respectively. HeLa cells showed no expression of CD36 on the cells surface (data not shown). Flow cytometric analysis of cell-phage binding test comprising of 10 randomly chosen clones showed clone B6 has the highest binding capacity towards CHO-CD36 cells compared to other cells (Fig. 1). Clone A6 and B11 showed a much lower CD36 binding ability. Similar trend can be seen in the binding of clone B6, A6 and B11 in U937 cells. Streptavidin phage control does not bind to any of the cells while the rest of the selected clones showed similar binding pattern to that of the Streptavidin phage control (data not shown). CD36-expressing cell lines were used to assess the phage ability to bind CD36 receptor on cells surface. CHO-CD36 cells are CD36-stably transfected cells and thus, express high levels of human CD36 (Hasler T, et. al. 1993) while U937 cells have been shown to express CD36 mRNA (Pietsch A, et.al. 1996). HeLa cells do not express CD36 at mRNA and protein levels (Ponten et al., 2009), thus, is a suitable negative control in assessing the specificity of the binding. Out of 24 clones identified, 10 were randomly chosen and tested for CD36 binding capacity. Since streptavidin phage was used as the phage control, its degree of binding was taken as the point of reference for non-specific binding. Hence, within the limit of flow cytometric assay, 3 clones have shown to be able to bind CD36 receptor on cells surface. In conclusion, Clone B6, A6 and B11 have different preferential binding pattern towards CD36 receptor on cells surface with B6 showing superior ability amongst the clones.
Acknowledgements
The authors would like to thank Ministry of Higher Education, Malaysia for funding this study under the Fundamental Research Grant Scheme (FRGS) 203/CIPPT/6711342 awarded to ISI.
Keywords:
Flow Cytometry,
CD36,
phage display,
Phage,
Cytometry
Conference:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting, Seberang Jaya, Penang, Malaysia, 17 Nov - 18 Nov, 2016.
Presentation Type:
Poster
Topic:
Gene Therapy, Reprogramming and Pluripotency
Citation:
Ayob
MS,
Yunus
MA,
Mohamed
R,
Arip
YM,
Das
KT and
Ismail
IS
(2016). CD36 binding ability of selected peptide phages identified by using phage display technique.
Front. Bioeng. Biotechnol.
Conference Abstract:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting.
doi: 10.3389/conf.FBIOE.2016.02.00006
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Received:
08 Dec 2016;
Published Online:
19 Dec 2016.
*
Correspondence:
Dr. Ida S Ismail, Universiti Sains Malaysia, Regenerative Medicine Cluster, Advanced Medical and Dental Institute (AMDI), Kepala Batas, Pulau Pinang, 13200, Malaysia, idashazrina@usm.my