Efficient identification of phosphatidylserine-binding proteins by ORF phage display.
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.
Caberoy
Nora B
NB
Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Miami, FL 33136, USA.
Zhou
Yixiong
Y
Alvarado
Gabriela
G
Fan
Xianqun
X
Li
Wei
W
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015723:Gene Library; D051379:Mice; D008894:Milk Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019151:Peptide Library; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D000954:Antigens, Surface; C490919:Mfge8 protein, mouse; D008894:Milk Proteins; D019151:Peptide Library; D010718:Phosphatidylserines; D011506:Proteins