DESIGN, EXPRESSION AND PURIFICATION OF VIAAT PROTEIN CONSTRUCT FOR CRYO-EM STRUCTURAL ANALYSIS
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Authors
M. Patapovich
Faculty of Biology, Belarusian State University, Nezavisimosti ave., 4, Minsk, 220030, Republic of Belarus
N. Lukashenko
Faculty of Biology, Belarusian State University, Nezavisimosti ave., 4, Minsk, 220030, Republic of Belarus
A. Yantsevich
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Academician Kuprevich str., 5/2, Minsk, 220084, Republic of Belarus
Abstract
In GABAergic neurons GABA is being transported into the synaptic vesicles by unique vesicular inhibitory amino acid transporter (VIAAT). Dysfunction of GABAergic system has been implicated into various diseases of neurological spectrum, making VIAAT a reasonable target for treatment. In order to develop specific modulators of VIAAT functioning, a well resolved three-dimensional structure of the transporter is needed. Such data is absent for the moment due to protein behavior in course of cryo-electron microscopy. In this work, interspecies VIAAT hybrid protein was designed in order to improve overall stability and introduce compatibility to the Fab fiducial marker, thus facilitating structure determination. Each variant consisted of the main part of drosophila VIAAT, in which the C-terminal part was replaced with the corresponding Fab binding region of human VIAAT. Interspecies parts of the hybrid protein were linked using a helical linker. Total of 9 hybrid protein variants were expressed in HEK293F cell line. Variants able to bind Fab were selected for further purification and applied to cryo-EM for structure determination. Three-dimensional data can contribute to the understanding of VIAAT structural organization which, in turn, facilitate the search for specific functional modulators as the perspective therapeutic agents for a wide variety of diseases.
Keywords
vesicular inhibitory amino acid transporter (VIAAT), γ-Aminobutyric acid (GABA), hybrid protein construct, neurotransmitters, fragment antigen binding (Fab), protein purification, cryo-EM
Article Details
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