Method for forming a reversible protein nanocluster using light in a cell

Patent metadata

Field	Value
Publication number	US-10604553-B2
Application number	US-201715618770-A
Country	US
Kind code	B2
Filing date	Jun 9, 2017
Priority date	Sep 30, 2011
Publication date	Mar 31, 2020
Grant date	Mar 31, 2020

Abstract

To efficiently analyze interaction and function between proteins, the present invention relates to a method for forming a light-induced protein nanocluster, comprising: an expression vector preparation step of preparing a first expression vector including polynucleotides coding a first fusion protein including a light-induced heterodimer-forming protein and a first self-assembly protein, and a second expression vector including polynucleotides coding a couple protein that forms a homodimer with said light-induced heterodimer-forming protein, or a second fusion protein including said couple protein and a second self-assembly protein; a transformed cell, tissue or individual preparation step of transforming cells, tissues or individuals using said first expression vector and second expression vector; and a light radiation step of radiating light having a wavelength for inducing the formation of heterodimer between said light-induced heterodimer-forming protein and said couple protein, to said transformed cells, tissue or individuals.

First claim

The invention claimed is:

1. A method for forming a protein nano-cluster, wherein the method comprises:
providing a first expression vector including a polynucleotide encoding a first fusion protein comprising a light-induced heterodimerized protein and a first self-assembled protein, and a second expression vector including a polynucleotide encoding a partner protein capable of forming a heterodimer with the light-induced heterodimerized protein, or a second fusion protein containing the partner protein and a second self-assembled protein; transforming a cell, a tissue or a subject with the first expression vector and the second expression vector;
irradiating light having wavelength capable of inducing heterodimerization between the light-induced heterodimerized protein and the partner protein to the cell, the tissue or the subject; and
forming a protein nano-cluster.

2. The method according to claim 1 , wherein the light-induced heterodimerized protein is CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein is CRY or PHR when the light-induced heterodimerized protein is CIB or CIBN, the partner protein is PIF when the light-induced heterodimerized protein is PhyB, the partner protein is GIFANTEA when the light-induced heterodimerized protein is FKF1, the partner protein is CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

3. The method according to claim 1 , wherein the first self-assembled protein and the second self-assembled protein is independently ferritin, virus capsid protein, ferritin-like protein, calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) or DsRed.

4. The method according to claim 1 , wherein at least one among the first fusion protein, the partner protein and the second fusion protein further comprises a fluorescent protein.

5. The method according to claim 4 , wherein the fluorescent protein is added to N-terminus or C-terminus of the first fusion protein, the partner protein and/or the second fusion protein or the fluorescent protein is inserted between the light-induced heterodimerized protein and the first self-assembled protein or between the partner protein and the second self-assembled protein.

6. The method according to claim 4 , wherein the fluorescent protein is green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein, cyan fluorescent protein (CFP), blue fluorescent protein (BFP), or tetracysteine fluorescent motif.

7. The method according to claim 5 , wherein the fluorescent protein is green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein, cyan fluorescent protein (CFP), blue fluorescent protein (BFP), or tetracysteine fluorescent motif.

Assignees

• Korea Advanced Inst Sci & Tech
• Korean Advanced Institute Of Science And Tech

Inventors

• Heo Won Do
• Lee Sang Kyu
• Park Hye Rim

Classifications

• C07K14/4728 Primary

  Calcium binding proteins, e.g. calmodulin · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• C12N9/93

  Ligases (6) · CPC title

• G01N33/582

  with fluorescent label · CPC title

• C07K14/325

  Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins · CPC title