Isolation of a Mycoplasma-specific binding peptide from an unbiased phage-displayed peptide library

Abstract

An important goal in medicine is the development of methods for cell-specific targeting of therapeutic molecules to pathogens or pathogen-infected cells. However, little progress has been made in cell-specific targeting of bacterially infected cells. Using a phage display approach, we have isolated a 20-mer peptide that binds to Mycoplasma arginini infected pancreatic beta-cells in tissue culture. This peptide binds to M. arginini infected beta-cells 200 times better than a control phage and is specific for the infected cells. Furthermore, transferring the M. arginini contamination to another cell line renders the newly infected cell line susceptible to peptide binding. Immunolocalization experiments suggest that the peptide is binding to M. arginini adhered to the cell surface. The free synthetic peptide retains its binding in the absence of the phage vehicle and tetramerization of the peptide increases its affinity for the infected cells. Efforts have been made to use this peptide to eliminate Mycoplasma from infected cell lines using ferromagnetic beads coated with the selected peptide. A ten-fold reduction of infection was accomplished with one fractionation via this approach. Our results suggest that this peptide, isolated from an unbiased selection, may be of utility for the detection and reduction of Mycoplasma infection in cultured cells. Furthermore, a general implication of our findings is that phage display methods may be useful for identifying peptides that target a broad array of other biological pathogens in a specific fashion.

Isolation of multiple cell-binding ligands from different phage displayed-peptide libraries

“…Selectivity and specificity determinations were performed as previously described using a control phage displaying the peptide sequence NQRGTELR-SPSVDLNKPGRH. The output/input ratio of the control phage is comparable to that of the whole library on H1299 cells indicating that this phage is a suitable control (De et al, 2005;McGuire et al, 2004aMcGuire et al, , 2004bOyama et al, 2003;Samli et al, 2005).…”

“…Panning phage-displayed peptide libraries on intact cells in culture, often referred to as biopanning, has proven to be successful for isolating cell-binding peptides (Barry et al, 1996;Brown, 2000;De et al, 2005;Ivanenkov et al, 1999aIvanenkov et al, , 1999bLandon and Deutscher, 2003;Mazzucchelli et al, 1999;McGuire et al, 2004aMcGuire et al, , 2004bSamli et al, 2005). Our laboratory and others have isolated cell-binding peptides, including cancer specific peptides, by this method (Bockman et al, 2005;Hong and Clayman, 2000;Landon and Deutscher, 2003;Oyama et al, 2003;Robinson et al, 2005;Romanov et al, 2001;Samoylova et al, 2002;Sioud, 2003, 2004;Zhang et al, 2001;Zitzmann et al, 2005).…”

Biopanning of Phage Displayed Peptide Libraries for the Isolation of Cell-Specific Ligands

“…The protocol is amenable to a variety of different cell types, including primary cells. To date, we have identified cell-specific peptides for many different cell types including cells of the immune system(2, 4), pancreatic β-cells(7), cardiac cells(3), tumor cells(5, 6), and pathogen-infected cells(8). Importantly, most peptides selected in this manner are active outside of context of the phage, retaining their cell-specificity and affinity.…”

B-cell Epitope Mapping of Helicobacter pylori Neutrophil-Activating Protein

“…The specificity of mAbs was identified by Western blot and immunohistochemical techniques. Finally, the mAbs were used as probes to immunoscreen a phage display random disulfide-constrained heptapeptide (C7C) library for obtaining mimicry B-cell epitopes [9,10].…”