Novel BRET combination for detection of rapamycin-induced protein dimerization using luciferase from fungus .

Bioluminescence resonance energy transfer (BRET) is one of the most promising approaches used for noninvasive imaging of protein-protein interactions . Recently, our team has discovered a genetically encodable bioluminescent system from the fungus and identified a novel luciferase that represents an imaging tool orthogonal to other luciferin-luciferase systems. We demonstrated the possibility of using the fungal luciferase as a new BRET donor by creating fused pairs with acceptor red fluorescent proteins, of which tdTomato provided the highest BRET efficiency.

Development of Noninvasive Activity-Based Protein Profiling-Bioluminescence Resonance Energy Transfer Platform Technology Enables Target Engagement Studies with Absolute Specificity in Living Systems.

Noninvasive, real-time, longitudinal imaging of protein functions in living systems with unprecedented specificity is one of the critical challenges of modern biomedical research. Toward that goal, here, we report a platform fusion technology called activity-based protein profiling-bioluminescence resonance energy transfer (ABPP-BRET). This method provides an opportunity to study the post-translational modification of a target protein in real time in living systems in a longitudinal manner.

Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase-Luciferin Pair.

This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase () from In vitro and in vivo assessments showed that human-codon-optimized () has significantly higher activity than native in various cancer cell types. The potential of in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), (RLuc), or nano-luciferase (NLuc).

In Vivo Assessment of Protein-Protein Interactions Using BRET Assay.

Proteins play an important part in almost all life activities and across all organisms. Proteins occasionally act on their own but rather fulfill most of their biological tasks by cooperating with other proteins or ligand molecules. The bioluminescence resonance energy transfer (BRET) assay serves to measure dynamic events such as protein-protein or protein-ligand interactions in vitro or in-vivo.

Direct knockdown of phospho-PTM targets mediated by TRIM21 can improve personalized treatment in breast cancer.

Purpose: In this work for the first time, we showed specific and direct knockdown of important oncogenic proteins of interest and their phospho-PTM targets in tripartite motif containing-21 (TRIM21) overexpressing breast cancer (BC) cells. We revealed the functional and therapeutic consequences of this protein knockdown approach called 'TRIM-ing'.

Methods: To target HER2, HER3, STAT3 or their activated forms, electroporation and puls-in transfection were standardized for mAb delivery in AU565 and MCF7 BC cell lines.