Japan Science and Technology Corporation

The University of Tokyo

Toyo Boseki Kabushiki Kaisha

Probex Inc.

Inter-university Research Institute Corporation National Institutes of Natural Sciences

Takeaki Ozawa

Yoshio Umezawa

Moritoshi Sato

Kenji Masuda

Shigeaki Nishii

Kenji Miura

Tasuku Okamoto

Naomi Misawa

Akira Kanno

The present invention intends to provide an assay system using split luciferase that has a remarkably high detection sensitivity. In an embodiment, binding of mutually binding first and second proteins is detected by preparing a first fusion protein comprising the first protein fused with a peptide having the amino acid sequence of amino acid SEQ ID NO: 1 and a second fusion protein comprising the second protein fused with a peptide having an amino acid sequence selected from the group consisting of amino acid SEQ ID NOS: 2 to 6, and allowing the first fusion protein to bind with the second fusion protein to form a complex, and detecting luminescence emitted from the complex.

Method for highly sensitive detection of protein-protein interaction

It is an object of the invention to provide novel approaches capable of detecting activated protease and also detecting protease activation on real time at a high sensitivity in a noninvasive manner. By the method for detecting activated protease of the invention, an indicator in a circular form comprising the C-half fragment of luciferase (Luc-C) and the N-half fragment of luciferase (Luc-N) linked together through a substrate peptide for a protease is introduced in an in vitro assay system or in cells. Upon digestion of the substrate peptide by the protease, Luc-N and Luc-C together reconstructs active luciferase, so that the activated protease can be detected by assaying the luminescence signal from the luciferase.

Activated protease indicator

As a versatile method of detecting and assaying intracellular protein phosphorylation and dephosphorylation that enables nondestructive monitoring as well as spatial and temporal analysis for living cells, animal bodies, plant bodies and the like, a probe for imaging protein phosphorylation and dephosphorylation, which comprises a tandem fusion unit composed of a substrate domain that contains a phosphorylation and dephosphorylation site, a linker sequence and a phosphorylation recognition domain, interposed between a donor chromophore and an acceptor chromophore that cause fluorescence resonance energy transfer, is used.

Probe for visualizing phosphorylation/dephosphorylation of protein and method of detecting and quantifying phosphorylation/dephosphorylation of protein

As a cGMP-visualizing probe capable of detecting and quantifying cGMP easily and accurately even in vivo and for a method of detecting and quantifying cGMP by using the same, a cGMP-visualizing probe comprising a polypeptide, which binds specifically to cGMP, and two chromophores having different fluorescence wavelengths each linked respectively to the two terminals of said polypeptide is provided.

Method of detection and quantification of cGMP by using a cGMP-visualizing probe

A probe for protein&#x2014;protein interaction analysis suitable for analyzing protein&#x2014;protein interactions of various proteins with high accuracy and in a simple manner and a method for analyzing interaction of two proteins by using the probe. With the probe, protein splicing is caused by protein&#x2014;protein interaction, and a physicochemically or biochemically detectable protein is regenerated.

Probe for analyzing protein&#x2014;protein interaction and method of analyzing protein&#x2014;protein interactions with the use of the same

cGMP-visualizing probe and a method of detecting and quantifying of cGMP by using the same

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Chiba, Japan

Takeaki Ozawa