title: Enhanced detection efficiency of genetically encoded tag allows the visualization of monomeric proteins by electron microscopy.
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.
authors.affiliation: Graduate School of Life Science,  University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297,  Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fukunaga
authors.forename: Ai
authors.initials: A
authors.lastname: Higashihara
authors.forename: Yuri
authors.initials: Y
authors.lastname: Nishino
authors.forename: Takuo
authors.initials: T
authors.lastname: Yasunaga
authors.forename: Mingyue
authors.initials: M
authors.lastname: Jin
authors.forename: Atsuo
authors.initials: A
authors.lastname: Miyazawa
mesh_terms: D002104:Cadmium / Q000378:metabolism; D002945:Cisplatin / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D010984:Platinum / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D002104:Cadmium; D049452:Green Fluorescent Proteins; D010984:Platinum; D008668:Metallothionein; D002945:Cisplatin