Takeaki Ozawa

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synec

The compartments of eukaryotic cells maintain a distinct protein composition to perform a variety of specialized functions. We developed a new method for identifying the proteins that are transported to the endoplasmic reticulum (ER) in living mammalian cells. The principle is based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803. Complementary DNA (cDNA) libraries fused to the N-terminal halves of

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTAdvances in Fluorescence and Bioluminescence ImagingTakeaki Ozawa*†, Hideaki Yoshimura†, and Sung Bae Kim‡View Author Information† Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan‡ Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan*E-mail: [email protected]. Pho

We describe a new method with general applicability for monitoring any protein-protein interaction in vivo. The principle is based on a protein splicing system, which involves a self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to formation of a new protein in which the exteins are linked directly by a peptide bond. As the exteins, split N- and C-terminal halves of enhanced green fluorescent protein (EGFP) were used. When a

Dysfunction of circadian clocks exacerbates various diseases, in part likely due to impaired stress resistance. It is unclear how circadian clock system responds toward critical stresses, to evoke life-protective adaptation. We identified a reactive oxygen species (ROS), H2O2 -responsive circadian pathway in mammals. Near-lethal doses of ROS-induced critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks, synergistically evoking protective responses for cell

In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which a

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can ove

We investigated the relationship between metal ion selective conformational changes of recoverin and its metal-bound coordination structures. Recoverin is a 23 kDa heterogeneously myristoylated Ca(2+)-binding protein that inhibits rhodopsin kinase. Upon accommodating two Ca(2+) ions, recoverin extrudes a myristoyl group and associates with the lipid bilayer membrane, which was monitored by the surface plasmon resonance (SPR) technique. Large changes in SPR signals were observed for Sr(2+), Ba(2+

In vivo bioluminescence imaging (BLI), which is based on luminescence emitted by the luciferase-luciferin reaction, has enabled continuous monitoring of various biochemical processes in living animals. Bright luminescence with a high signal-to-background ratio, ideally red or near-infrared light as the emission maximum, is necessary for in vivo animal experiments. Various attempts have been undertaken to achieve this goal, including genetic engineering of luciferase, chemical modulation of lucif

Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK)-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P) in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a nov

Forensic science is an important field of analytical chemistry where vibrational spectroscopy, in particular Fourier transform infrared spectroscopy and Raman spectroscopy, present advantages as they have a nondestructive nature, high selectivity, and no need for sample preparation. Herein, we demonstrate a method for determination of donor sex, based on attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy of dry urine traces. Trace body fluid evidence is of special im