Clonable Gold Label

About

Background: 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. Applications: 1. For electron microscopy studies, MT-tag allows efficient labeling of intracellular molecules with gold without compromising the samples with detergent treatment. 2. Metallothionein also binds other heavy metal atoms such as mercury and silver. MT-tagging technology can be used as an affinity-purification means in conjunction with an immobilized affinity column charged with metal ions such as cadmium. Advantages: • Provides a method for labeling and purification of proteins. This technology can be easily incorporated into cloning kits and protein purification kits • Electron-dense gold clusters form directly onto the MT-tags of targeted proteins. Therefore, this labeling method does not require demembranating samples and provides more accurate and complete protein localization than is possible with gold-nanocluster-conjugated secondary antibodies. • MT-tag is one-forth the size of GFP, hence posing 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. • Since MT-tag can be used for affinity purification, this invention provides a means to isolate the very same protein, for further analyses. It may even allow co-purification of specific organelle markers, which can then be used to verify the intracellular location of the targeted proteins. Technology: 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, like green fluorescent protein, can be cloned onto a 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.