Concatenated metallothionein as a clonable gold label for electron microscopy.
Localization of proteins in cells or complexes using electron microscopy has mainly relied upon the use of heavy metal clusters, which can be difficult to direct to sites of interest. For this reason, we would like to develop a clonable tag analogous to the clonable fluorescent tags common to light microscopy. Instead of fluorescing, such a tag would initiate formation of a heavy metal cluster. To test the feasibility of such a tag, we exploited the metal-binding protein, metallothionein (MT). We created a chimeric protein by fusing one or two copies of the MT gene to the gene for maltose binding protein. These chimeric proteins bound many gold atoms, with a conservative value of 16 gold atoms per copy of metallothionein. Visualization of gold-labeled fusion proteins by scanning electron microscopy required one copy of metallothionein while transmission electron microscopy required two copies. Images of frozen-hydrated samples of simple complexes made with anti-MBP antibodies hint at the usefulness of this method.
Mercogliano
Christopher P
CP
Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
DeRosier
David J
DJ
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006046:Gold / Q000737:chemistry*; D008668:Metallothionein / Q000737:chemistry* / Q000302:isolation & purification; D046529:Microscopy, Electron, Transmission; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D006046:Gold; D008668:Metallothionein
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