Method and kit for analyzing protein-protein interaction using nanocluster formation

Patent metadata

Field	Value
Publication number	US-9784747-B2
Application number	US-201214234243-A
Country	US
Kind code	B2
Filing date	Jul 23, 2012
Priority date	Jul 22, 2011
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

For efficient analysis of a protein-protein interaction, the present disclosure provides a kit for analyzing a protein-protein interaction, the kit including: a 1 st expression vector including a 1 st polynucleotide and a multi-cloning site, wherein, the 1 st polynucleotide is operably linked to a promoter and encodes a 1 st fusion protein having a 1 st fluorescence protein and a 1 st self-assembly protein, and the multi-cloning site is a site where a polynucleotide encoding a bait protein may be operably linked to the polynucleotide encoding the 1 st fusion protein; and a 2 nd expression vector including a 2 nd polynucleotide and a multi-cloning site, wherein, the 2 nd polynucleotide is operably linked to a promoter and encodes a 2 nd fusion protein having a 2 nd fluorescence protein and a 2 nd self-assembly protein, and the multi-cloning site is a site where a polynucleotide encoding a prey protein may be operably linked to the polynucleotide encoding the 2 nd fusion protein.

First claim

The invention claimed is:

1. A kit for analyzing a protein-protein interaction, comprising:
a 1 st expression vector including a 1 st gene construct having a 1 st polynucleotide which is operably linked to a promoter and encodes a 1 st fusion protein having a bait protein, a 1 st fluorescence protein and a 1 st self-assembly protein; and
a 2 nd expression vector including a 2 nd gene construct having a 2 nd polynucleotide which is operably linked to a promoter, and encodes a 2 nd fusion protein having a prey protein, a 2 nd fluorescence protein and a 2 nd self-assembly protein,
wherein the 1 st fluorescence protein and the 2 nd fluorescence protein emit light having diffferent wavelengths from each other; the 1 st self-assembly protein and the 2 nd self-assembly protein do not interact with each other and the 1 st self-assembly protein and the 2 nd self-assembly protein form nanoparticles by self-assembly, respectively; and the 1 st fluorescence protein or the 2 nd fluorescence protein is omittable when either the 1 st fluorescence protein or the 2 nd fluorescence protein is DsRed, and
wherein nanoclusters are formed when the nanoparticies interact with each other.

2. The kit of claim 1 , wherein the 1 st fluorescence protein is a green fluorescent protein ((GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), ECFP, TagCFP, DsRed or a tetracystein fluorescent motif.

3. The kit of claim 1 , wherein the 1 st self-assembly protein is ferritin, a virus capsid protein, magnetosome, calmodulin kinase IIα (CaMKIIα) or DsRed.

4. The kit of claim 3 , wherein the virus capsid protein is a cowpea chlorotic mottle virus (CCMV) capsid protein, a norwalk virus capsid protein, a SV40 major capsid protein, or a papilloma virus capsid protein.

5. The kit of claim 1 , wherein the 2 nd fluorescence protein is a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), ECFP, TagCFP, DsRed or a tetracystein fluorescent motif.

6. The kit of claim 1 , wherein the 2 nd self-assembly protein is ferritin, a virus capsid protein, maenetosome, calmodulin kinase IIα (CaMKIIα) or DsRed.

7. The kit of claim 6 , wherein the virus capsid protein is a CCMV (cowpea chlorotic mottle virus) capsid protein, a norwalk virus capsid protein, a SV40 major capsid protein, or a papilloma virus capsid protein.

8. The kit of claim 1 , wherein the promoter is a eukaryotic promoter.

9. The kit of claim 1 , wherein the promoter is a eukaryotic promoter.

10. A kit for analyzing a protein-protein interaction, comprising:
a 1 st expression vector including a 1 st polynucleotide and a multi-cloning site, wherein, the 1 st polynucleotide is operably linked to a promoter and encodes a 1 st fusion protein having a 1 st fluorescence protein and a 1 st self assembly protein, and the multi-cloning site is a site where a polynucleotide encoding a bait protein may be operably linked to the polynucleotide encoding the 1 st fusion protein; and
a 2 nd expression vector including a 2 1 polynucleotide and a multi-cloning site, wherein, the 2 nd polynucleotide is operably linked to a promoter and encodes a 2 nd fusion protein having a 2 1 fluorescence protein and a 2 nd self-assembly protein, and the multi-cloning site is a site where a polynucleotide encoding a prey protein may be operably linked to the polynucleotide encoding the 2 nd fusion protein,
wherein the 1 st fluorescence protein and the 2 nd fluorescence protein emit light having diffferent wavelengths from each other; the 1 st self-assembly protein and the 2 nd self-assembly protein do not interact with each other and the 1 st self-assembly protein and the 2 nd self-assembly protein form nanoparticles by self-assembly, respectively; and the 1 st fluorescence protein or the 2 nd fluorescence protein is omittable when either the 1 st fluorescence protein or the 2 nd fluorescence protein is DsRed, and wherein nanociusters are formed when the nanoparticles interact with each other.

11. The kit of claim 2 , wherein the 1 st fluorescence protein is a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), ECFP, TacCFP, DsRed or a tetracystein fluorescent motif.

12. The kit of claim 10 , wherein the 1 st self-assembly protein is ferritin, a virus capsid protein, magnetosome, calmodulin kinase IIα (CaMKIIα) or DsRed.

13. The kit of claim 12 , wherein the virus capsid protein is a cowpea chlorotic mottle virus (CCMV) capsid protein, a nor alk virus capsid protein, a SV40 major capsid protein, or a papilloma virus capsid protein.

14. The kit of claim 10 , wherein the 2 nd fluorescence protein is a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), ECFP, TagCFP DsRed or a tetracystein fluorescent motif.

15. The kit of claim 10 , wherein the 2 nd self-assembly protein is ferritin, a virus capsid protein, magnetosome, calmodulin kinase IIα (CaMKIIα) or DsRed.

16. The kit of claim 15 , wherein the virus capsid protein is a CCMV chlorotic mottle virus) capsid protein, a norwalk virus capsid protein, a SV40 major capsid protein, or a papilloma virus capsid protein.

17. A method for analyzing a protein-protein interaction, comprising:
transfecting a cell with a 1 st expression vector including a 1 st gene construct and a 2 nd expression vector including a 2 nd gene construct, wherein the 1 st gene construct having a 1 st polynucleotide which is operably linked to a promoter and encodes a 1 st fusion protein having a bait protein, a 1 st fluorescence protein and a 1 st self-assembly protein, and the 2 nd gene construct having a 2 nd polynucleotide which is operably linked to a promoter and encodes a 2 nd fusion protein having a prey protein, a 2 nd fluorescence protein and a 2 nd self-assembly protein;
culturing the transfected cell; and
observing distribution and intensity of fluorescence in the cell with a fluorescence microscopy,
wherein the 1 st fluorescence protein and the 2 nd fluorescence protein emit light having different wavelengths from each other; the 1 st self-assembly protein and the 2 nd self-assembly protein do not interact with each other and form nanoparticles by self-assembly, respectively; and the 1 st fluorescence protein or the 2 nd fluorescence protein is omittable when either the 1 st fluorescence protein or the 2 nd fluorescence protein is DsRed, and
wherein nanoclusters are formed when the nanoparticles interact with each other.

18. The method of claim 17 , wherein the 1 st fluorescence protein is a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), ECFP, TagCFP, DsRed or a tetracystein fluorescent motif.

19. The method of claim 17 , wherein the 1 st self-assembly protein is ferritin, a virus capsid protein, magnetosome, calmodulin kinase IIα (CaMKIIα) or DsRed.

20. The method of claim 19 , wherein the virus capsid protein is a cowpea chlorotic mottle virus (CCMV) capsid protein, a nor alk virus capsid protein, a SV40 major capsid protein, or a papilloma virus capsid protein.

Assignees

• Heo Won Do
• Kim Sun Chang
• Lee Sang Kyu
• Korea Advanced Inst Sci & Tech

Inventors

• Heo Won Do
• Kim Sun Chang
• Lee Sang Kyu

Classifications

• G01N33/582

  with fluorescent label · CPC title

• C07K2319/735

  containing a domain for self-assembly, e.g. a viral coat protein (includes phage display) · CPC title

• G01N33/6845 Primary

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• C12N15/79

  Vectors or expression systems specially adapted for eukaryotic hosts · CPC title

• G01N2333/91205

  Phosphotransferases in general · CPC title