title: Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates.
abstract: We report the preparation and performance of TEM grids bearing stabilized nonfouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly(ethylene glycol) 2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir-Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(5-amido-1-carboxypentyl)iminodiacetic acid]polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10',12'-diynoyl)-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE)-in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication. These conditions were designed to limit nonspecific protein adsorption onto the stabilized lipid coating by favoring the formation of a mPEG350 brush layer below a flexible, mushroom conformation of NTA-PEG2000 at low surface density to enable specific immobilization and random orientation of the protein target on the EM grid. These grids were then used to capture His6-T7 bacteriophage and RplL from cell lysates, as well as purified His8-green fluorescent protein (GFP) and nanodisc solubilized maltose transporter, His6-MalFGK2. Our findings indicate that TEM grid supported, polymerized NTA lipid monolayers are capable of capturing His-tag protein targets in a manner that controls their areal densities, while efficiently blocking nonspecific adsorption and limiting film degradation, even upon prolonged detergent exposure.
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Benjamin
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Wright
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Seok-Hee
authors.initials: SH
authors.lastname: Hyun
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Kyle
authors.initials: K
authors.lastname: Krynski
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Guimei
authors.initials: G
authors.lastname: Yu
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Ruchika
authors.initials: R
authors.lastname: Bajaj
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Fei
authors.initials: F
authors.lastname: Guo
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Cynthia V
authors.initials: CV
authors.lastname: Stauffacher
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Wen
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Chemistry and ‡Department of Biological Sciences, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: David H
authors.initials: DH
authors.lastname: Thompson
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics; D000327:Adsorption; D017123:Bacteriophage T7 / Q000737:chemistry; D002457:Cell Extracts / Q000737:chemistry; D020285:Cryoelectron Microscopy / Q000295:instrumentation*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation; D009571:Nitrilotriacetic Acid / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics; D013499:Surface Properties
chemical_list: C519184:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol 2000); D018528:ATP-Binding Cassette Transporters; D002457:Cell Extracts; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; C521492:MPEG350; D009842:Oligopeptides; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C461599:maltose transport system, E coli; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D006639:Histidine; C016700:ribosomal protein L7-L12; C000595210:polyethylene glycol 2000; D009571:Nitrilotriacetic Acid