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Application of Nonc...
Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli
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- Kipper, Kalle (author)
- Uppsala universitet,Institutionen för cell- och molekylärbiologi,Science for Life Laboratory, SciLifeLab
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- Lundius, Ebba G. (author)
- Uppsala universitet,Science for Life Laboratory, SciLifeLab,Institutionen för cell- och molekylärbiologi
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- Ćurić, Vladimir, 1981- (author)
- Uppsala universitet,Institutionen för cell- och molekylärbiologi,Science for Life Laboratory, SciLifeLab
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- Nikic, Ivana (author)
- European Mol Biol Lab, Cell Biol & Biophys Unit, Struct & Computat Biol Unit, D-69117 Heidelberg, Germany.
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- Wiessler, Manfred (author)
- Deutsch Krebsforschungszentrum, Biol Chem, D-69120 Heidelberg, Germany.
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- Lemke, Edward A. (author)
- European Mol Biol Lab, Cell Biol & Biophys Unit, Struct & Computat Biol Unit, D-69117 Heidelberg, Germany.
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- Elf, Johan (author)
- Uppsala universitet,Molekylär systembiologi,Science for Life Laboratory, SciLifeLab
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(creator_code:org_t)
- 2016-11-23
- 2017
- English.
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In: ACS Photonics. - : AMER CHEMICAL SOC. - 2330-4022. ; 6:2, s. 233-255
- Related links:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Subject headings
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- Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Keyword
- noncanonical amino acid
- tetrazine
- recoded E. coli
- in vivo fluorescence labeling
- OmpC
- single particle tracking
Publication and Content Type
- ref (subject category)
- art (subject category)
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