Cloneable Nanoparticles for Electron Microscopy

At a Glance

Researchers at Colorado State University have developed metal-reducing enzyme (MRE) tags that enable precise protein localization in advanced imaging. These tags produce nanoparticles in cells, allowing seamless integration into electron, x-ray, and optical microscopy. This groundbreaking technology offers unparalleled imaging resolution and fluorescent stability for studying biological systems.

Background

Biological imaging tools like green fluorescent protein (GFP) revolutionized cellular research by enabling protein localization with fluorescence microscopy. However, achieving atomic-level resolution and combining electron and optical imaging in a single system has remained a challenge. Metal-reducing enzymes provide a novel solution by producing localized nanoparticles that visualize proteins in high-resolution imaging techniques.

Overview

This technology leverages the unique properties of a Glutathione Reductase-like Metalloid Reductase (GSHRMR) enzyme . These MRE’s alone catalyze the in situ formation of metal nanoparticles, including selenium, tellurium, cadmium, and zinc. This allows precise control over particle size and retention. Unlike traditional methods, these tags act as a biological scaffold, seamlessly integrating into electron microscopy, cryo-EM, and optical imaging workflows.

The MREs demonstrate key advantages, such as high substrate specificity and the ability to increase metal tolerance tenfold in engineered E. coli. Quantum dots formed by these enzymes exhibit fluorescence that resists bleaching, enabling extended optical observations. The versatility of the MRE tags allows for the creation of a kit that facilitates easy incorporation of these tags into various research workflows, enabling the localization and study of proteins at resolutions previously unattainable.

Figure 1. Electron tomographic reconstructions of P. moraviensis Stanleyae revealing SeNPs within the cytoplasm of the cell (Panels A and B). Panel C shows a native PAGE gel with lanes from left to right: 1) GSHRMR soaked in a solution of 50 mM HEPES, 1 mM NADPH and 5 mM Na2SeO3, 2) Coomassie stained GSHRMR, 3) prestained protein ladder.

Figure 2. TEM images and overlaying EDS maps of E. coli grown with a short induction with IPTG of the GSHRMR-12 concatemer tag and a short incubation time in selenite. Se, indicated in red and seems to be localizing at the pinch point of the cells as the division progresses (Panels A thru C) indicating the presence of active EM tagged FtsZ with SeNPs at the Z-ring to cause the separation of the two cells.

Figure 3. The approach: A metalloid reducing enzymatic gene will be cloned into a gene of interest and when expressed will produce a fusion protein of the target protein and the redox active tag. · Incubation with the metal salt precursors leads to the reduction or the precursors and subsequent nucleation and growth of an inorganic NP.

Benefits

Precise Size Control: Nanoparticles as small as 5 nanometers can be reliably produced.
Simplified Production: A single enzyme catalyzes the entire process, making it easier to replicate.
Improved Stability: Particles are stable without the need for coatings or shells.
Produces stable quantum dots resistant to fluorescence bleaching.
Facilitates the synthesis of diverse metal nanoparticles.

Applications

Advanced biological imaging for research and diagnostics.
Contrast markers for electron microscopy.
High-resolution protein localization in cellular and tissue studies.
Quantum dot synthesis for optical labeling.
Development of new nanotechnology materials.

Publications

Nemeth et al (2018) “Metalloid reductase of Pseudomonas moravenis Stanleyae conveys nanoparticle mediated metalloid tolerance.” ACS Omega. https://pubs.acs.org/doi/10.1021/acsomega.8b00826

Ni, et al (2015) “Progress Toward Cloneable Inorganic Nanoparticles.” Nanoscale. https://doi.org/10.1039/c5nr04097c

Last Updated: February 2025

Opportunity

Available for Exclusive Licensing

IP Status

US 17/266,734

Inventors

Chris Ackerson
Zach Butz
Richard Nemeth
Ryan Riskowski
Kanda Borgonon

Reference Number

19-012

Licensing Manager

Jessy McGowan
Jessy.McGowan@colostate.edu
970-491-7100