Efficient identification of phosphatidylserine-binding proteins by ORF phage display.

Nora B Caberoy, Yixiong Zhou, Gabriela Alvarado, Xianqun Fan, Wei Li

Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Miami, FL 33136, USA.

Biochemical and biophysical research communications 2009 Aug 14

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To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in approximately 300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

Citation

Nora B Caberoy, Yixiong Zhou, Gabriela Alvarado, Xianqun Fan, Wei Li. Efficient identification of phosphatidylserine-binding proteins by ORF phage display. Biochemical and biophysical research communications. 2009 Aug 14;386(1):197-201