Enhanced detection efficiency of genetically encoded tag allows the visualization of monomeric proteins by electron microscopy

J Electron Microsc (Tokyo). 2012 Aug;61(4):229-36. doi: 10.1093/jmicro/dfs043. Epub 2012 Apr 16.

Abstract

A cadmium-binding, genetically encoded protein tag, consisting of three repeats of metallothionein (3MT), can be used in electron microscopy for the visualization of multimeric- but not monomeric-tagged proteins due to insufficient electron density in monomeric proteins. Here, we present a technique for detecting monomeric 3MT-tagged green fluorescent protein (GFP-3MT) using a platinum compound to intensify the electron density. Substitution of cadmium by platinum as a result of incubating purified cadmium-binding 3MT-tagged GFP (GFP-Cd-3MT) with cis-diamminedichloroplatinum(II) (cisDDP) was assessed by a UV absorption band centered at 284 nm thereby indicating platinum-sulfhydryl bonds. The incubation time and the concentration of cisDDP to reach maximal absorption were 2 h and 36-fold molar equivalent of cisDDP, respectively. GFP-Pt-3MT isolated by gel filtration chromatography contained 29 platinum atoms per single GFP-3MT molecule. Electron-dense particles were observed in a GFP-Pt-3MT sample by electron microscopy without negative staining. Further image processing and image analysis demonstrated that particles with higher density relative to their surroundings were detectable in both GFP-Cd-3MT and GFP-Pt-3MT samples. These results demonstrate that replacement of cadmium with platinum, together with proper image analyses, improve detection efficiency and enable the visualization of 3MT-tagged monomeric protein by electron microscopy.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cadmium / metabolism
  • Cisplatin / metabolism
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics*
  • Green Fluorescent Proteins / metabolism
  • Metallothionein / chemistry
  • Metallothionein / genetics*
  • Metallothionein / metabolism
  • Microscopy, Electron, Transmission / methods*
  • Platinum / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Recombinant Fusion Proteins
  • Cadmium
  • Green Fluorescent Proteins
  • Platinum
  • Metallothionein
  • Cisplatin