Protein-Protein Interactions as a Tool for Site-Specific Labeling of Proteins
Allows for site-specific labeling and can be used with longer proteins.Can be performed in simple batch-mode.No need for time-consuming two-step chromatography to separate singly-labeled proteins from non- or doubly-labeled side-products.Can be used when sequential labeling does not work.Allows for 3-color labeling of recombinantly expressed proteins.
Site-specific labeling of a polypeptide chain with a unique donor/acceptor fluorophore pair for FRET monitoring of protein folding reactions.Rapid optimization of dye pairs (i.e., to optimize the FRET-efficiencies for protein folding studies).Possible development into a general method for site-specific protein labeling.
UCLA investigators have developed a novel method of utilizing protein-protein interactions to site-specifically label recombinantly expressed multi-Cys proteins. An engineered Cys side chain can be physically protected from conjugation with extrinsic fluorophores by the burial of surface area in the protein-protein interaction. Meanwhile, a second Cys remains solvent-exposed after complex formation and can be selectively labeled in the pre-assembled complex. By utilizing the surface area burial in protein-protein interactions, this invention can be performed in simple batch-mode without a need to separate singly-labeled proteins from non- or doubly-labeled side-products. Thus, it allows for simple site-specific labeling of recombinant proteins for FRET-based single molecule studies.
Patent Number: US8084226B2
Application Number: US2006490240A
Inventor: Weiss, Shimon | Jäger, Marcus
Priority Date: 21 Jul 2005
Priority Number: US8084226B2
Application Date: 21 Jul 2006
Publication Date: 27 Dec 2011
IPC Current: C12P000100 | A61K003800
US Class: 435041 | 530345
Assignee Applicant: The Regents of the University of California
Title: Method for site-specific protein modifications
Usefulness: Method for site-specific protein modifications
Summary: The methods are useful for site-specific modification of protein molecules, for site-specific protein labeling, and for producing a product comprising protein molecules modified or labeled according to the methods given (claimed).
生物医学
医学影像
8084226
Background Related Materials Protein-protein interactions as a tool for site-specific labeling of proteins Additional Technologies by these Inventors Tech ID/UC Case 21720/2005-314-0 Related Cases 2005-314-0
However, a critical component in this experiment is the ability to label a polypeptide chain with a unique donor/acceptor fluorophore pair in a controlled and site-specific way. Current methods of labeling polypeptide chains have certain limitations. Chemical synthesis can be exploited to facilitate site-specific 2-color labeling, but run into difficulties for 3-color labeling or for proteins longer than 100 amino acids. Recombinant expression of proteins using Cys residues can be used with longer proteins, but is not strictly site-specific. This can lead to unwanted sample heterogeneity and preclude 3- or multi-color FRET experiments. Finally, a novel method of site-specific incorporation of non-natural amino acids into proteins in vivo using genetically modified orthogonal t-RNA/t-RNA synthetase pairs is powerful but not yet broadly available to the scientific community. Thus, there is a need for a new method of labeling a polypeptide chain without all of the limitations.
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