scholarly journals Versatile labeling and detection of endogenous proteins using tag-assisted split enzyme complementation

2020 ◽  
Author(s):  
Suraj Makhija ◽  
David Brown ◽  
Struan Bourke ◽  
Yina Wang ◽  
Shuqin Zhou ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
Protein Interaction ◽  
Genome Engineering ◽  
Small Peptide ◽  
Protein Protein Interaction ◽  
Recent Advances ◽  
Cell Enrichment ◽  
Peptide Tags ◽  
Endogenous Proteins

AbstractRecent advances in genome engineering have expanded our capabilities to study proteins in their natural states. In particular, the ease and scalability of knocking-in small peptide tags has enabled high throughput tagging and analysis of endogenous proteins. To improve enrichment capacities and expand the functionality of knock-ins using short tags, we developed the tag-assisted split enzyme complementation (TASEC) approach, which uses two orthogonal small peptide tags and their cognate binders to conditionally drive complementation of a split enzyme upon labeled protein expression. Using this approach, we have engineered and optimized the tag-assisted split HaloTag complementation system (TA-splitHalo) and demonstrated its versatile applications in improving the efficiency of knock-in cell enrichment, detection of protein-protein interaction, and isolation of biallelic gene edited cells through multiplexing.

Protein-Protein Interaction (PPI) Network: Recent Advances in Drug Discovery

2017 ◽  
Vol 18 (1) ◽  
pp. 5-10 ◽  
Author(s):  
Alexiou Athanasios ◽  
Vairaktarakis Charalampos ◽  
Tsiamis Vasileios ◽  
Ghulam Ashraf
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Ppi Network ◽  
Protein Protein Interaction ◽  
Recent Advances

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

scholarly journals Split-TurboID enables contact-dependent proximity labeling in cells

2020 ◽  
Author(s):  
Kelvin F. Cho ◽  
Tess C. Branon ◽  
Sanjana Rajeev ◽  
Tanya Svinkina ◽  
Namrata D. Udeshi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interaction ◽  
Proteomic Analysis ◽  
Protein Protein Interaction ◽  
Conventional Fractionation ◽  
Contact Sites ◽  
Biochemical Fractionation ◽  
Versatile Tool ◽  
Endogenous Proteins ◽  
In Cells

AbstractProximity labeling (PL) catalyzed by promiscuous enzymes such as TurboID have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum (ER)-mitochondria contact sites, reconstituted TurboID catalyzed spatially-restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to ER-mitochondria contacts. We validated eight novel candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially-specific proximity labeling in cells.

scholarly journals Megadock-Web: An Integrated Database of High-Throughput Structure-Based Protein-Protein Interaction Predictions

2019 ◽  
Vol 116 (3) ◽  
pp. 563a
Author(s):  
Masahito Ohue ◽  
Takanori Hayashi ◽  
Yuri Matsuzaki ◽  
Keisuke Yanagisawa ◽  
Yutaka Akiyama
Keyword(s):  
High Throughput ◽  
Protein Interaction ◽  
Protein Protein Interaction

Recent advances in the techniques of protein-protein interaction study

2013 ◽  
Vol 35 (11) ◽  
pp. 1274-1282
Author(s):  
Ming-Qiang WANG ◽  
Jin-Xia WU ◽  
Yu-Hong ZHANG ◽  
Ning HAN ◽  
Hong-Wu BIAN ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Interaction Study ◽  
Protein Protein Interaction ◽  
Recent Advances
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