Saltar al contenido
Merck

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.

Proceedings of the National Academy of Sciences of the United States of America (2014-10-15)
Daniel S Liu, Lucas G Nivón, Florian Richter, Peter J Goldman, Thomas J Deerinck, Jennifer Z Yao, Douglas Richardson, William S Phipps, Anne Z Ye, Mark H Ellisman, Catherine L Drennan, David Baker, Alice Y Ting
RESUMEN

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
Cloruro de sodio, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
L-Glutamina, meets USP testing specifications, suitable for cell culture, 99.0-101.0%, from non-animal source
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Cloruro de sodio, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
L-Glutamina, ReagentPlus®, ≥99% (HPLC)
SAFC
Sodium chloride solution, 5 M
SAFC
L-Glutamina
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Cloruro de sodio, 99.999% trace metals basis
Sigma-Aldrich
Cloruro de sodio, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, 2 M in H2O
Sigma-Aldrich
Magnesium chloride, BioReagent, suitable for insect cell culture, ≥97.0%
Sigma-Aldrich
L-Glutamina, BioUltra, ≥99.5% (NT)
Sigma-Aldrich
Resorufin, 95%
Sigma-Aldrich
Magnesium chloride solution, PCR Reagent, 25 mM MgCI2 solution for PCR
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.9% trace metals basis
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative
Sigma-Aldrich
Cloruro de sodio, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Glutaraldehyde solution, 50 wt. % in H2O
Sigma-Aldrich
Glutaraldehyde solution, Grade II, 25% in H2O
Sigma-Aldrich
Glutaric dialdehyde solution, 50 wt. % in H2O, FCC
Sigma-Aldrich
Cloruro de sodio, BioPerformance Certified, ≥99% (titration), suitable for insect cell culture, suitable for plant cell culture
Sigma-Aldrich
Cloruro de sodio, AnhydroBeads, −10 mesh, 99.999% trace metals basis
Sigma-Aldrich
L-Glutamina
Sigma-Aldrich
Cloruro de sodio, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl