Clonable Gold Label for Electron Microscopy
Clonable Gold Label for EM
The Situation: Current methods for labeling proteins for electron microscopy (EM) use gold clusters attached to specific tags on proteins. The fraction of tags labeled with a gold cluster is small, and cell membranes must be removed so that gold clusters can enter cells. Non-specific attachment of gold clusters can cause misinterpretation of images.
Our Solution: We developed a novel clonable protein tag that allows gold atoms to form a cluster directly onto the protein tag, thereby circumventing problems associated with introducing preformed-gold clusters into cells and with non-specific labeling. This clonable label also provides a way to purify protein by affinity columns.
Transmission electron microscopy (TEM), which can resolve cellular structures down to Angstrom levels, has been a great source of information for a wide range of biological studies. Clonable labeling tags for light microscopy, such as green fluorescent protein, have revolutionized biology, but few such agents have been developed for TEM. Gold nanoclusters are traditionally used to label biological structures for electron microscopy studies; however, introducing preformed heavy metal clusters into cells is an inefficient labeling process, and detergent treatment of tissue samples necessary for metal cluster penetration can cause damage to samples, resulting in experimental artifacts. We have developed a clonable labeling method for TEM in which a small metal-binding protein, metallothionein (MT), is used as a fusion tag for labeling proteins. By incubating cells in a solution with gold-containing compounds, gold atoms can diffuse across cell membranes and assemble directly onto the MT-tag of targeted protein. This may circumvent the problems associated with introducing preformed gold clusters into cells.
- Efficient gold-labeling of intracellular molecules in EM applications without compromising the samples with detergent treatments.
- Allows affinity-purification of tagged protein with immobilized affinity columns charged with cadmium ions.
- Provides a method for labeling and purification of proteins. This technology can be easily incorporated into cloning kits and protein purification kits.
- No need to de-membranate samples.
- Superb accuracy and sensitivity in protein localization than is possible with gold-nanocluster-conjugated secondary antibodies.
- Compact MT-tag poses fewer risks for steric hindrance and folding problems to targeted proteins.
- Metallothionein is present in many organisms. Species-specific MT-expression vectors can be tailored-made for different biological systems, thereby optimizing protein expression and avoiding codon-usage problems caused by a “generic” tag.
- Affinity purification capacity of MT-tag can be used for isolating the very same protein for further analyses. Co-purification of specific organelle markers can help to verify the intracellular locations of targeted proteins.
This invention provides compositions and methods for heavy atom labeling of target proteins using a clonable metallothionein (MT) tag. Metallothioneins constitute a super family of low molecular weight, cysteine-rich proteins that bind to various metal ions to protect cells from heavy metal toxicity. This invention applies the metal-binding characteristic of metallothionein for the development of a novel protein labeling method for electron microscopy. The MT-tag, with molecular weight only one-forth of that of green fluorescent protein (GFP), can be cloned onto any protein of interest. The gold label permits visualization of target protein by electron microscopy, but unlike traditional gold labeling methods, electron-dense gold clusters assemble directly onto the MT-tag from monomeric gold and does not require forceful introduction of preformed gold clusters into cells. In addition to gold, MT-tag permits protein labeling by other heavy metals, such as silver and mercury. Furthermore, MT-tag provides means for affinity purification of protein. The inventors have successfully demonstrated that proteins labeled with MT can be efficiently purified on immobilized metal affinity chromatography columns charged with cadmium ions.
- Patent pending in United States
- Pending international patent application
Chris Mercogliano and David DeRosier.
To discuss this technology with a licensing officer, please call Irene Abrams at (781)-736-2176 or email email@example.com and ask about record ID: 2004-0202.