A genetically encoded metallothionein tag enabling efficient protein detection by electron microscopy.
The observation of biological materials by transmission electron microscopy (TEM) frequently requires the use of negative staining and/or immuno-gold labeling to visualize or identify the specimen, but these techniques are limited. In contrast, genetic labeling with green fluorescent protein (GFP) and its homologs have led to rapid advances in the observation of proteins of interest in cells by light microscopy (LM). These fluorescent tags allow for the visualization of dynamic processes in live cells without the use of secondary reagents. Here, we report the development of an artificial metalloprotein fusion protein expressed in Escherichia coli grown in Cd(2+)-containing medium that allows for efficient protein detection by TEM without additional staining steps. We linked the subunits of the bacterial 14-mer protein GroEL with three repeats of metallothionein (3MT). The 3MT-fused GroEL (GroEL-14(3MT)) was successfully expressed in E. coli, and the purified protein included 250 cadmium atoms per molecule on average. Cd(2+)-bound GroEL-14(3MT) was detected by TEM in the absence of negative staining on a carbon grid, and the particle densities of GroEL-14(3MT) were much greater than those of untagged GroEL in vitreous ice. Taken together, our data indicate that the 3MT tag provides a promising means of allowing the identification of oligomeric proteins isolated from cells in the absence of other detection techniques.
Nishino
Yuri
Y
Bio-multisome Research Team, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Yasunaga
Takuo
T
Miyazawa
Atsuo
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure*; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D016324:Sequence Tagged Sites
D018834:Chaperonin 60; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D008668:Metallothionein