title: 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.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Benjamin
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Wright
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Bolton
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Seok-Hee
authors.initials: SH
authors.lastname: Hyun
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Kyle
authors.initials: K
authors.lastname: Krynski
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Mahima
authors.initials: M
authors.lastname: Grover
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Guimei
authors.initials: G
authors.lastname: Yu
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Guo
authors.affiliation: Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Tamara L
authors.initials: TL
authors.lastname: Kinzer-Ursem
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.|Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, USA.|Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Thompson
mesh_terms: D010129:4-Aminobenzoic Acid / Q000737:chemistry; D017123:Bacteriophage T7 / Q000737:chemistry / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D006108:Graphite / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D008567:Membranes, Artificial; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation*; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010087:Oxides / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C097388:2-N,N-bis(carboxymethyl)lysine; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D008567:Membranes, Artificial; D009842:Oligopeptides; D010087:Oxides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D006108:Graphite; D008239:Lysine; D010129:4-Aminobenzoic Acid