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US 2025/0145676 A1Published application
Functional expression of ectopic tmc1 and tmc2 in heterologous cells
AssigneeThe Hong Kong University of Science and Technology
InventorsPingbo HUANG, Yixuan CHEN
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Abstract
The subject invention pertains methods for the ectopic expression of a transmembrane protein lacking plasma membrane expression in a heterologous cell line. In embodiments, transmembrane proteins Fyn-TMC1 and/or Fyn-TMC2 are fused to a membrane-anchoring motif, a Fyn lipidation tag, which provides a signal for myristoylation and palmitoylation, allowing further research on these proteins, including but not limited to their function, structure, and drug screening.
Claims12
A method for the ectopic expression of a transmembrane protein, the method comprising:
- (a) obtaining an ectopic transmembrane protein lacking plasma membrane expression in a heterologous cell line;
- (b) expressing the ectopic transmembrane protein on the plasma membrane of a heterologous cell by fusing a tag at the N-terminus, wherein the tag comprises a Fyn lipidation tag or other motif that serves as a signal for myristoylation and palmitoylation modifications;
- and
- (c) performing an assay,
- wherein the assay comprises:
- (i) a transmembrane protein functional assay or transmembrane protein structural assay;
- or
- (ii) a medium throughput assay for screening specific high-affinity drugs interacting with the transmembrane protein, a high throughput assay for screening specific high-affinity drugs interacting with the transmembrane protein, or a basal intracellular calcium signal assay;
- (iii) a calcium imaging assay for determining changes in basal intracellular calcium signal after addition of a pharmaceutical compound, a drug binding affinity to the transmembrane protein, or measuring activity of the transmembrane protein after exposure of novel aminoglycoside antibiotics utilizing patch-clamp;
- or
- (iv) a drug screening assay for high-affinity drugs.
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