Ligand/binding partner bio-labeling systems
First Claim
1. A method of detecting a target protein in a mammalian host cell, wherein the mammalian host cell expresses an endogenous functional dihydrofolate reductase gene, comprising:
- (a) preparing a nucleic acid construct comprising a promoter element operatively linked to a nucleic acid encoding the target protein and a nucleic acid encoding a bacterial dihydrofolate reductase;
(b) introducing the nucleic acid construct into a host cell;
(c) culturing the host cell under conditions such that a target protein/dihydrofolate reductase fusion protein is expressed;
(d) introducing, into the host cell cultured according to step (c), a detectably labeled trimethoprim; and
(e) detecting the presence of the target protein by detecting labeled trimethoprim bound to the fusion protein.
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Abstract
The present invention relates to methods and compositions which utilize binding relationships between detector molecules and their ligands for labeling molecules of interest in vivo. A “target” molecule is linked to a detector molecule (such as a protein or a nucleic acid) and the function and/or location of the target molecule is monitored by binding the detector molecule its ligand, which carries a detectable label. In particular embodiments of the invention, an intracellular fusion protein comprising a target protein and a detector protein is bound to a membrane-permeable, fluorescently-labeled ligand of the detector protein, thereby providing an adjunct or alternative to Green Fluorescent Protein.
2 Citations
3 Claims
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1. A method of detecting a target protein in a mammalian host cell, wherein the mammalian host cell expresses an endogenous functional dihydrofolate reductase gene, comprising:
- (a) preparing a nucleic acid construct comprising a promoter element operatively linked to a nucleic acid encoding the target protein and a nucleic acid encoding a bacterial dihydrofolate reductase;
(b) introducing the nucleic acid construct into a host cell;
(c) culturing the host cell under conditions such that a target protein/dihydrofolate reductase fusion protein is expressed;
(d) introducing, into the host cell cultured according to step (c), a detectably labeled trimethoprim; and
(e) detecting the presence of the target protein by detecting labeled trimethoprim bound to the fusion protein. - View Dependent Claims (2, 3)
- (a) preparing a nucleic acid construct comprising a promoter element operatively linked to a nucleic acid encoding the target protein and a nucleic acid encoding a bacterial dihydrofolate reductase;
Specification