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Title: Protein- protein interaction detection system using fluorescent protein microdomains

Abstract

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

Inventors:
 [1];  [2]
  1. Santa Fe, NM
  2. Los Alamos, NM
Issue Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1014181
Patent Number(s):
7666606
Application Number:
US Patent Application 11/295,368
Assignee:
Los Alamos National Security, LLC (Los Alamos, NM)
Patent Classifications (CPCs):
G - PHYSICS G01 - MEASURING G01N - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
C - CHEMISTRY C40 - COMBINATORIAL TECHNOLOGY C40B - COMBINATORIAL CHEMISTRY
DOE Contract Number:  
W-7405-ENG-36
Resource Type:
Patent
Country of Publication:
United States
Language:
English

Citation Formats

Waldo, Geoffrey S, and Cabantous, Stephanie. Protein- protein interaction detection system using fluorescent protein microdomains. United States: N. p., 2010. Web.
Waldo, Geoffrey S, & Cabantous, Stephanie. Protein- protein interaction detection system using fluorescent protein microdomains. United States.
Waldo, Geoffrey S, and Cabantous, Stephanie. Tue . "Protein- protein interaction detection system using fluorescent protein microdomains". United States. https://www.osti.gov/servlets/purl/1014181.
@article{osti_1014181,
title = {Protein- protein interaction detection system using fluorescent protein microdomains},
author = {Waldo, Geoffrey S and Cabantous, Stephanie},
abstractNote = {The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2010},
month = {2}
}

Works referenced in this record:

Circularly permuted green fluorescent proteins engineered to sense Ca2+
journal, March 2001


Combinatorial Marking of Cells and Organelles with Reconstituted Fluorescent Proteins
journal, October 2004


A Fluorescent Indicator for Detecting Protein−Protein Interactions in Vivo Based on Protein Splicing
journal, November 2000


Seeing what was unseen: New analytical methods for molecular imaging
journal, January 2003


Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation
journal, April 2002


Antiparallel Leucine Zipper-Directed Protein Reassembly:  Application to the Green Fluorescent Protein
journal, June 2000