Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide

Abstract

We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with Nα, Nα-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.

General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM

“…For challenging systems and dynamic complexes, the typical path of overexpression, biochemicalscale purification and concentration can be problematic, either because the system cannot be reconstituted, or aggregation may occur. Perhaps even more significant is the disruption of protein structure and protein-protein interactions that can occur when exposed to the air water interface 5 . During the formation of the very thin vitreous ice films (often <50nm) required for high resolution imaging, the very high surface area to volume ratio dramatically increases the probability of exposure to the denaturing interface.…”

The His-tag as a decoy modulating preferred orientation in cryoEM

“…One of the strategies for changing interactions of macromolecules with a support, and with the water-air interface, is to chemically modify the molecule itself without changing the chemical properties of the support. Affinity tags chemically modify a protein and have been used in sample preparation for cryoEM in the context of affinity purification (Wang, Liu, et al, 2020, Benjamin, Wright, Bolton, et al, 2016, but as we observed, tag presence can modify interactions with the water-air interface even without the use of affinity grids.…”

“…They include using surfactants (Chen et al, 2019, Glaeser et al, 2016, Frederik et al, 1989 to saturate the surface at the water-air interface or the surface of the support (Russo & Passmore, 2014), a graphene oxide or graphene supports to prevent interactions the water-air interface (Pantelic et al, 2010, Pantelic et al, 2011, Wang, Liu, et al, 2020. These supports may also be chemically modified to promote specific, high-affinity interactions with particles (Llaguno et al, 2014, Kelly et al, 2010a, Kelly et al, 2008, Kelly et al, 2010b, Han et al, 2012, Benjamin, Wright, Bolton, et al, 2016, Crucifix et al, 2004, Wang, Liu, et al, 2020, Yeates et al, 2020. Finally, new, fast approaches for depositing samples on a grid have been developed (Arnold et al, 2017, Schmidli et al, 2018, Dandey et al, 2020, Ravelli et al, 2019.…”

Cryo-EM: beyond the microscope

“…This has successfully been used for the high resolution 3D reconstruction of an icosahedral virus [63]. A similar approach, using Ni-NTA modified grids to capture His-tagged proteins, has also been used to perform “on-grid” purification [64,65], including for smaller protein complexes (Figure 3a). However, both of these techniques can also have limitations.…”