Intercellular labeling of ligand-receptor interactions
AssigneeWhitehead Institute for Biomedical Research
InventorsGiulia Pasqual, Gabriel Victora
Life of this patent
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Abstract
An sortase-mediated intercellular labeling method allowing for tracking ligand-receptor interaction both in vitro and in vivo; and uses thereof for tracking molecule interactions both in vitro and in vivo, identifying modulators of ligand-receptor interaction, identifying potential binding partners of a protein of interest, identifying B cells expressing high affinity B cell receptors to antigens, and identifying the antigen to which a T cell of interest binds.
Claims31
An intercellular labeling method comprising:
- (i) providing a first cell expressing a first polypeptide on the surface of the first cell, the first polypeptide comprising a sortase acceptor peptide, which is located at the N-terminus of the first polypeptide;
- (ii) providing a second cell expressing a second polypeptide on the surface of the second cell, the second polypeptide comprising a sortase or an active fragment thereof;
- and
- (iii) contacting the first cell with the second cell in the presence of a sortase substrate comprising a sortase recognition sequence, wherein the sortase substrate is associated with a detectable label;
- wherein upon interaction between the first cell and the second cell, the sortase or the active fragment thereof links the sortase substrate to the first polypeptide, thereby labeling the first cell expressing the first polypeptide.
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34-59. (canceled)
A non-human animal, comprising:
- (i) a first cell expressing a first polypeptide on its surface, the first polypeptide comprising a sortase acceptor peptide, which is located at the N-terminus of the first polypeptide;
- (ii) a second cell expressing a second polypeptide on its surface, the second polypeptide comprising a sortase, or
- (iii) both (i) and (ii).
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A nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising a sortase and a member of a ligand-receptor pair.
87-119. (canceled)
A method for identifying a binding partner of a protein of interest, the method comprising:
- (i) providing a first population of cells expressing a plurality of polypeptides, each of which expresses a sortase acceptor peptide and a candidate protein;
- (ii) providing a second population of cells expressing a sortase or an active fragment thereof, and the protein of interest;
- (iii) contacting the first population of cells with the second population of cells in the presence of a sortase substrate comprising a sortase recognition sequence, wherein the peptide is associated with a detectable label;
- (iv) detecting labeling of cells in the first population of cells;
- and
- (iv) identifying a binding partner of the protein of interest, wherein a candidate protein is a binding partner of the protein of interest, if the cell that expresses a polypeptide comprising the candidate protein is labeled in step (iii).
121-135. (canceled)
Dependencies are inferred from the claim text.