Structural Analysis of HEV-3 ORF2 Protein

A total of 3 μL of purified HEV-3 ORF2 110–610_6his recombinant protein was adsorbed onto glow discharged Carbon-Formvar-coated copper grids for 60 s. The grids were then washed by floating on water droplets, followed by staining with 2% (w/v) uranyl acetate (UA) for 20 s. Particles were imaged using an FEI Tecnai G2 20 (FEI, Hillsboro, OR, USA) electron microscope operating at 200 kV. An Integra Prima microscope and Nova SPM software (NT-MDT, Moscow, Russia) were used for atomic force microscopy. Scanning was performed in semi-contact mode, using gold cantilever NSG01 (NT-MDT). The protein sample was applied to a sapphire substrate coated with mica and dried at room temperature. PBS was used as a negative control. Cryo-EM grids were prepared as described by Byrne et al., 2019 [49 (link)].

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Takova K., Koynarski T., Minkov G., Toneva V., Mardanova E., Ravin N., Lukov G.L, & Zahmanova G. (2021). Development and Optimization of an Enzyme Immunoassay to Detect Serum Antibodies against the Hepatitis E Virus in Pigs, Using Plant-Derived ORF2 Recombinant Protein. Vaccines, 9(9), 991.

Publication 2021

Tecnai G2 20 Integra Prima microscope Nova SPM software gold cantilever NSG01

Atomic force microscopy Carbon Copper Electron microscope Formvar Gold Mica Microscope Protein Recombinant protein Sapphire Uranyl acetate

Corresponding Organization : Center of Plant Systems Biology and Biotechnology

Other organizations : Trakia University, Russian Academy of Sciences, Brigham Young University–Hawaii

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Variable analysis

independent variables

Amount of purified HEV-3 ORF2 110–610_6his recombinant protein adsorbed onto the grids (3 μL)

dependent variables

Imaging of the adsorbed particles using an electron microscope
Imaging of the protein sample using atomic force microscopy

control variables

Glow discharged Carbon-Formvar-coated copper grids used for protein adsorption
Washing of the grids by floating on water droplets
Staining of the grids with 2% (w/v) uranyl acetate for 20 s
Microscope settings (FEI Tecnai G2 20 electron microscope operating at 200 kV; Integra Prima microscope and Nova SPM software for atomic force microscopy)
Scanning mode (semi-contact mode) and cantilever (gold cantilever NSG01) for atomic force microscopy
Protein sample applied to a sapphire substrate coated with mica and dried at room temperature for atomic force microscopy
PBS used as a negative control for atomic force microscopy

controls

Positive control: Not specified
Negative control: PBS used for atomic force microscopy

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