Development of Clonable Electron-Dense Labels for Electr

Office Of The Director, Nih

Investigators

Abstract

We are investigating the feasibility of developing clonable electron-dense labels that can be visualized in three-dimensional reconstructions of subcellular organelles obtained by electron tomography. Currently, electron tomography is often limited by uncertainty in determining which proteins of interest correspond to specific features in the reconstruction. Clonable labels could potentially localize those proteins, by analogy to green fluorescent protein in optical microscopy. Bacterial ferritin, an iron storage protein that accumulates a dense iron oxide nanoparticle in its hollow core, is a promising candidate. A modified ferritin gene from Listeria innocua was cloned into Escherichia coli so that the inserted DNA coded for the 12 ferritin subunits of the ferritin connected by flexible linker sequences. The modified gene has been overexpressed and experiments are in progress to determine whether the engineered protein folds properly. The next step is to join the ferritin gene construct to the gene of interest in E. coli to form a fusion protein that can be expressed in the E. coli. Novel electron tomography techniques based on scanning transmission electron microscopy and energy-filtering transmission electron microscopy are being developed to help visualize the electron-dense particles of Listeria innocua ferritin, each of which contains a few hundred iron atoms.