title: Concatenated metallothionein as a clonable gold label for electron microscopy.
abstract: 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.
authors: Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
authors: Christopher P
authors: CP
authors: Mercogliano
authors: David J
authors: DJ
authors: DeRosier
mesh_terms: 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
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D006046:Gold; D008668:Metallothionein
references: J Histochem Cytochem. 2000 Apr;48(4):471-80
references: 10727288
references: J Struct Biol. 2006 Nov;156(2):334-41
references: 16857386
references: J Bacteriol. 2001 Nov;183(21):6404-12
references: 11591685
references: J Am Chem Soc. 2002 May 29;124(21):6063-76
references: 12022841
references: EMBO J. 2002 Nov 15;21(22):5969-78
references: 12426369
references: J Biol Inorg Chem. 2003 Nov;8(8):831-42
references: 14505073
references: J Inorg Biochem. 1980 Jul;12(4):293-306
references: 7411139
references: Science. 1982 Oct 15;218(4569):290-1
references: 7123234
references: J Biol Chem. 1983 Nov 10;258(21):13063-9
references: 6630220
references: J Biol Chem. 1985 May 10;260(9):5342-50
references: 3988757
references: Biochemistry. 1985 Apr 9;24(8):1977-86
references: 4016095
references: Methods Enzymol. 1991;205:252-63
references: 1779785
references: J Struct Biol. 1996 Jan-Feb;116(1):17-24
references: 8742718
references: Biochemistry. 1996 Nov 5;35(44):13937-45
references: 8909291
references: J Struct Biol. 1999 Sep;127(2):106-12
references: 10527899
references: J Mol Biol. 2006 Jan 13;355(2):211-23
references: 16305802
references: Nat Rev Mol Cell Biol. 2006 Mar;7(3):225-30
references: 16482091
references: Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14245-50
references: 11087814