Phage Display

A major goal in medicine is the development of methods for cell-specific targeting of therapeutic agents to diseased cells. My group at the Center for Biomedical Inventions (CBI) has developed phage display technologies to identify peptides that mediate binding and uptake into target cells. Using our approach, we have identified targeting peptides for 25 different cell types. The protocol involves panning of the library with intact cells. After incubation with the phage library, the cells are subjected to a series of stringent washes designed to remove unbound phage as well as phage that are bound to the surface of the cell. The cell lysates are used to infect E. coli, enabling phage retrieval and amplification. Importantly, the selected peptides show unprecedented cell-specificities, even among similar cells. This is remarkable when one considers that no counter selections against other cell types were performed. The origin of this specificity is still unknown, however it suggests that the peptides are binding to receptors present at vastly different levels on the cell surface in different cell types or are recognizing distinct cellular landscapes that vary from cell type to cell type. We have shown that the peptides can home to desired cell types in vivo. In addition, we have developed methodology for removing the peptide from the phage while retaining its affinity and specificity for the desired cell type.

Because our targeting peptides are identified from a library-based assay, no prior knowledge of the cellular receptors expressed on the cell surface of the target cell is required. This allows for the identification of cellular receptors that may not have been previously considered as therapeutic targets or may not have been identified. It also allows us to map interesting surface features of a cell. We are employing biochemical techniques to identify the receptors. This project involves development of new methodologies to identify protein-protein interactions. In particular, we are developing new cross-linking methodologies that allow for specific protein-protein interactions to be detected. The strategy involves incubating the targeting-peptides with cells, then using chemical cross-linking to trap the non-covalent interactions between the peptide and its putative receptor covalently. Our choice of a chemical cross-linking strategy was largely the result of the development of a new type of cross-linking reagents by researchers at the CBI that are much faster and more efficient than standard reagents. These reagents mediate efficient oxidative coupling of closely associated polypeptides in a reaction that requires only a few seconds. The cross-linked species are then isolated by SDS-PAGE, and the protein identified using mass spectrometry.

Figure I

 

Figure II

Development of Efficient and Highly Specific Cross-Linking

CGH-Ni(II) complex activated by addition of peracid

Ru(II)bp₃²⁺+ APS activated by 452nm light

Advantages over traditional cross-linking:

• High yields: 20-80%
• High specific: only proteins that interact in solution are cross-linked
• Rapid rates of reaction allowing for short-lived interactions to be trapped
• Tyrosines are the primary amino acid targets, however, other cross-linked products can be formed
• The cross-linking reagents can easily be attached to peptides

Figure III

 

Related References:

Amini, Frank; Kodadek, Thomas; Brown, Kathlynn C. (2002) “ Protein Affinity Labeling Mediated by a Genetically-Encoded Tag” Angewante Chem. 41, 356-359.

Person, Maria D.; Brown, Kathlynn C.; Mahrus, Sami; Craik, Charles S.;Burlingame, Alma L. (2001) “Mass Spectrometric Identification of a Novel Cross-linked Adduct in the GGH-ecotin D137Y.” Protein Science 10, 1549-1562.

Brown, Kathlynn C. and Kodadek, Thomas “Protein Cross-Linking Mediated by Metal Ion Complexes”in "Probing of Proteins by Metal Ion and Their Low-Molecular-Weight Complexes", Vol. 38 of 'Metal Ions in Biological Systems', A. Sigel and H. Sigel, eds. M. Dekker, Inc., New York, 2001, pages 351-384.

Brown, Kathlynn C.(2000) “New Approaches for Cell-Specific Targeting Identification of Cell-Selective Peptides from Combinatorial Libraries” Curr. Opin. Chem. Biol. 4, 16-21.

Kim, Kunghee; Fancy, David A.; Kodadek, Thomas (1999) “Photo-induced protein cross-linking mediated by metalloporphyrins” J. Am. Chem. Soc., 121, 11896.

Fancy, David .A. and Kodadek, Thomas (1999) “ Chemistry for the analysis of protein-protein interactions: Rapid and eficient cross-linking triggered by long wavelength light” Proc. Natl. Acad. Sci USA, 96, 6020.

Brown, K.C. Yu, Z.; Burlingame, A.L.; Craik, C.S.; (1998) “Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein” Biochemistry, 37, 4397.

Brown, K.C.; Yang, S.-H.; Kodadek, T.; (1995) “ Highly specific oxidative protein cross-linking mediated by a nickel peptide complex” Biochemistry, 34, 4733.