Journal ArticleDOI
Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells
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TLDR
This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.Abstract:
Recombinant proteins containing four cysteines at the i , i + 1, i + 4, and i + 5 positions of an α helix were fluorescently labeled in living cells by extracellular administration of 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein. This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.read more
Citations
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Fluorescent amino acids as versatile building blocks for chemical biology
TL;DR: This Review covers recent advances in the design and synthesis of fluorescent amino acids with bespoke optical properties for different applications in biological studies, as well as to investigate biological processes in cells by means of optical imaging.
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Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro.
TL;DR: This work has developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation.
Journal ArticleDOI
Illuminating the life of GPCRs.
TL;DR: Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented.
Journal ArticleDOI
Programmed loading and rapid purification of engineered bacterial microcompartment shells.
TL;DR: Shell proteins are functionalised to facilitate facile purification and enable cargo encapsulation via covalent linkage and the generalizability of CAP and EnCo will enable their application to functionally diverse microcompartment systems to facilitate both characterization of natural functions and the development of bespoke shells for selectively compartmentalizing proteins.
Journal ArticleDOI
A Fluorogenic TMP-Tag for High Signal-to-Background Intracellular Live Cell Imaging
Chaoran Jing,Virginia W. Cornish +1 more
TL;DR: The results suggest that the fluorogenic TMP-tag can significantly impact high-resolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering.
References
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Journal ArticleDOI
The green fluorescent protein
TL;DR: In just three years, the green fluorescent protein from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology.
Journal ArticleDOI
Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.
François Denizot,Rita Lang +1 more
TL;DR: The reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays.
Journal ArticleDOI
Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin
Atsushi Miyawaki,Juan Llopis,Roger Heim,J. Michael McCaffery,Joseph A. Adams,Mitsuhiko Ikura,Mitsuhiko Ikura,Roger Y. Tsien +7 more
TL;DR: New fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations are constructed and dubbed ‘cameleons’.
Journal ArticleDOI
Crystal structure of the Aequorea victoria green fluorescent protein.
TL;DR: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products.
Journal ArticleDOI
Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer
Roger Heim,Roger Y. Tsien +1 more
TL;DR: The results demonstrate that the production of more and better GFP variants is possible and worthwhile, and facilitates multicolor imaging of differential gene expression, protein localization or cell fate.
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