Patent ID: 8293500

Claim:
A method of identifying a cellular polypeptide to which a viral polypeptide binds, said method comprising: (a) contacting (i) a fraction or a supernatant obtained from a plurality of cells, and (ii) a fusion protein comprising a viral virulence polypeptide moiety fused to an affinity tag moiety, to permit the viral virulence polypeptide moiety to interact with a polypeptide associated with the cell fraction or the cell supernatant, to provide a fusion protein:cellular polypeptide complex, wherein the affinity tag moiety comprises a first polypeptide tag and a second polypeptide tag wherein the first polypeptide tag comprises a protein C-tag and the second polypeptide tag comprises a streptavidin binding peptide; (b) contacting the fusion protein:cellular polypeptide complex with a cognate ligand capable of interacting with the affinity tag, to permit formation of a cognate ligand:fusion protein:cellular polypeptide complex; (c) isolating the fusion protein:cellular polypeptide complex from the cognate ligand:fusion protein:cellular polypeptide complex by affinity purification; (d) subsequent to affinity purification, (A) determining the amino acid sequence of the cellular polypeptide or (B) determining the amino acid sequence of at least one fragment of the cellular polypeptide, comprising: (i) cleaving the fusion protein:cellular polypeptide complex of step (c) with a protease to generate a plurality of polypeptide fragments of the cellular polypeptide; (ii) determining the amino acid sequence of at least one polypeptide fragment of the cellular polypeptide, wherein the fragment comprises at least eight amino acids; and (iii) comparing the amino acid sequence of the at least one polypeptide fragment with the amino acid sequence of a known cellular polypeptide; and (e) identifying the cell type of a cell that comprises the cellular polypeptide to which the viral virulence polypeptide binds, said step comprising: (i) contacting the fusion protein and a biological sample comprising a plurality of cells, to permit the viral virulence polypeptide moiety of the fusion protein to interact with the cells; (ii) determining the presence or absence of binding of the fusion protein to the cells; (iii) isolating the cells to which the fusion protein binds; and (iv) characterizing the cells, and therefrom determining the cell type of the cell that comprises a cellular polypeptide to which the viral virulence polypeptide binds.