Related Experiment Video
Updated: Jul 10, 2026
09:22
Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure
Published on: April 28, 2023
A genetically encoded metallothionein tag enabling efficient protein detection by electron microscopy
Yuri Nishino1, Takuo Yasunaga, Atsuo Miyazawa
1Bio-multisome Research Team, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Journal of Electron Microscopy
|October 31, 2007
Summary
Researchers developed a novel metalloprotein tag for transmission electron microscopy (TEM). This artificial tag enables direct visualization of proteins, overcoming limitations of traditional staining methods in biological imaging.
Area of Science:
- Biophysics
- Structural Biology
- Microscopy
Background:
- Transmission electron microscopy (TEM) often requires negative staining or immuno-gold labeling for biological specimen visualization.
- These traditional TEM techniques have inherent limitations in identifying specific proteins.
- Genetic labeling with fluorescent proteins (e.g., GFP) has advanced live-cell imaging but is not directly applicable to TEM.
Purpose of the Study:
- To develop a novel method for efficient protein detection in TEM without additional staining.
- To create an artificial metalloprotein fusion tag for direct visualization of proteins by TEM.
- To assess the utility of a metallothionein (MT) tag for enhancing protein visibility in TEM.
Main Methods:
- Engineered a fusion protein by linking bacterial GroEL subunits with three repeats of metallothionein (3MT).
- Expressed the 3MT-fused GroEL (GroEL-14(3MT)) in Escherichia coli cultured in cadmium (Cd(2+))-containing medium.
- Analyzed purified GroEL-14(3MT) using TEM, with and without negative staining, and compared particle densities with untagged GroEL.
Main Results:
- Successfully expressed and purified GroEL-14(3MT), with an average of 250 cadmium atoms per molecule.
- Detected Cd(2+)-bound GroEL-14(3MT) directly by TEM on a carbon grid, without negative staining.
- Observed significantly higher particle densities for GroEL-14(3MT) compared to untagged GroEL in vitreous ice.
Conclusions:
- The 3MT tag facilitates efficient protein detection by TEM, eliminating the need for secondary staining or labeling.
- This metalloprotein fusion strategy offers a promising approach for identifying oligomeric proteins directly from cellular samples.
- The developed tag enhances protein visibility in TEM, potentially simplifying structural biology workflows.
Related Concept Videos
01:24Tagging and Fusion Proteins
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
01:19Translocation of Proteins into the Mitochondria
Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
01:20Immunogold Electron Microscopy
Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.