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A genetically encoded aldehyde for rapid protein labelling.

ABSTRACT: Using a mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pair, 3-formyl-phenylalanine is genetically incorporated into proteins at amber mutation sites in Escherichia coli. This non-canonical amino acid readily reacts with hydroxylamine dyes, leading to rapid and site-selective protein labelling.

SUBMITTER: Tuley A

PROVIDER: S-EPMC4062584 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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A genetically encoded aldehyde for rapid protein labelling.

Tuley Alfred A Lee Yan-Jiun YJ Wu Bo B Wang Zhiyong U ZU Liu Wenshe R WR

Chemical communications (Cambridge, England) 20140701 56

Using a mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pair, 3-formyl-phenylalanine is genetically incorporated into proteins at amber mutation sites in Escherichia coli. This non-canonical amino acid readily reacts with hydroxylamine dyes, leading to rapid and site-selective protein labelling. ...[more]

PMID: 24756176

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Project description:BackgroundThe dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure.MethodsFor this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression.ResultsThe biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice.ConclusionsWe have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.

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A genetically encoded aldehyde for rapid protein labelling.

Publications

A genetically encoded aldehyde for rapid protein labelling.

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