Compositions and methods for monitoring transmembrane trafficking

Patent metadata

Field	Value
Publication number	US-9738921-B2
Application number	US-201514800352-A
Country	US
Kind code	B2
Filing date	Jul 15, 2015
Priority date	Nov 23, 2011
Publication date	Aug 22, 2017
Grant date	Aug 22, 2017

Abstract

Provided herein are compositions and methods for monitoring the movement of analytes and/or cellular components across biological membranes (e.g., cell surface internalization). In particular, reporter constructs are provided, the transmembrane movement of which (e.g., by endocytosis) is monitored by methods described herein.

First claim

We claim:

1. A method of detecting endocytosis comprising:
a) expressing a fusion protein in a cell comprising a cell membrane, wherein said fusion protein comprises a cell-surface receptor protein and a reporter protein, and said expressing results in the co-expression of the cell surface receptor protein and the reporter protein as a single fusion protein in the cell, wherein the reporter protein is displayed extracellularly when the cell-surface receptor is incorporated into the cell membrane of the cell;
b) delivering a reporter substrate to the cell, wherein an association between the reporter protein and reporter substrate produces a detectable signal when the association occurs extracellularly and an altered signal when the association occurs intracellularly; and
c) monitoring said detectable signal and said altered signal, wherein a transition from said detectable signal to said altered signal indicates endocytosis of the fusion protein.

2. The method of detecting endocytosis of claim 1 , wherein monitoring of said detectable signal comprises:
i) inducing endocytosis of said fusion protein; and
ii) detecting said detectable signal and said altered signal after inducing endocytosis.

3. The method of detecting endocytosis of claim 1 , wherein monitoring of said detectable signal comprises:
i) detecting said signal prior to endocytosis;
ii) allowing endocytosis of said fusion protein;
iii) detecting said signal following endocytosis; and
iv) comparing said signal from step (i) to said signal from step (iii) to detect endocytosis.

4. The method of claim 1 , wherein said reporter protein comprises a luciferase enzyme.

5. The method of claim 4 , wherein the luciferase enzyme is a beetle or Oplophorus luciferase enzyme or a mutant or variant thereof.

6. The method of claim 1 , wherein endocytosis results in movement of said reporter protein from an extracellular space into an endosome.

7. The method of claim 6 , wherein said signal is detectably altered within said endosome compared to the extracellular space.

8. The method of claim 6 , wherein said signal is reduced within said endosome compared to the extracellular space.

9. The method of claim 1 , wherein said reporter substrate is a luciferin, luciferin derivative, coelenterazine or coelenterazine derivative.

10. The method of claim 3 , further comprising a step between steps (i) and (ii) of triggering endocytosis.

Assignees

• Promega Corp

Inventors

• Binkowski Brock F
• Cong Mei
• Robers Matthew B

Classifications

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title

• G01N33/567

  utilising isolate of tissue or organ as binding agent · CPC title

• C12Q1/66 Primary

  involving luciferase · CPC title

• G01N33/58

  involving labelled substances (G01N33/53 takes precedence) · CPC title