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Bacterial Production and Structural Study of Human Neuromodulator Lynx2

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Abstract

The human protein Lynx2 belongs to the Ly6/uPAR family of “three-finger” proteins and is associated with the cell membrane by the glycosylphosphatidylinositol (GPI) anchor. Lynx2 is expressed in the brain areas responsible for anxiety level control: prefrontal cortex, basolateral tonsil, hippocampus, and medial dorsal nucleus of the thalamus; it probably participates in anxiety regulation by interaction with α4β2 nicotinic acetylcholine receptor. Currently, the nature of the Lynx2 interaction with the receptor is unknown. We developed a bacterial expression system for production of a Lynx2 water-soluble domain (ws-Lynx2). The protein was obtained in the form of E. coli cytoplasmic inclusion bodies, with subsequent solubilization under denaturing conditions and renaturation. Production of milligram quantities of the 13С-15N-labeled variant of ws-Lynx2 made it possible to characterize the secondary structure of this protein by means of heteronuclear NMR spectroscopy. It was shown that ws-Lynx2 has structure typical for three-finger proteins from the Ly6/uPAR family with some unique features, such as helical elements in the first and third loops. Development of the effective recombinant production system opens up new horizons for further structural and functional studies of Lynx2.

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Funding

The work was supported by the Russian Science Foundation (project no. 19-74-20176).

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Authors and Affiliations

  1. Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Moscow, 117997, Russia

    A. S. Paramonov, M. A. Shulepko, M. V. Kocharovskaya, A. E. Alenkin, A. O. Evdokimova, P. I. Akentiev, Z. O. Shenkarev, M. P. Kirpichnikov & E. N. Lyukmanova

  2. Moscow Institute of Physics and Technology, Dolgoprudnyi, Moscow oblast, 141700, Russia

    M. V. Kocharovskaya, A. O. Evdokimova, P. I. Akentiev & E. N. Lyukmanova

  3. Faculty of Biology, Moscow State University, Moscow, 119192, Russia

    A. E. Alenkin & M. P. Kirpichnikov

Authors
  1. A. S. Paramonov
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  2. M. A. Shulepko
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  3. M. V. Kocharovskaya
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  4. A. E. Alenkin
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  5. A. O. Evdokimova
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  6. P. I. Akentiev
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  7. Z. O. Shenkarev
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  8. M. P. Kirpichnikov
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  9. E. N. Lyukmanova
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Corresponding author

Correspondence to E. N. Lyukmanova.

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The article contains no studies involving human or animals as subjects of the study.

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Translated by N. Onishchenko

Abbreviations: DTT, dithiothreitol (1,4-bis(sulfanyl)butane-2,3-diol); IPTG, isopropyl β-D-1-thiogalactopyranoside; NOE, nuclear Overhauser effect; GPI, glycosylphosphatidylinositol; GSH and GSSG, reduced and oxidized forms of glutathione; nAChR, nicotinic acetylcholine receptor.

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Paramonov, A.S., Shulepko, M.A., Kocharovskaya, M.V. et al. Bacterial Production and Structural Study of Human Neuromodulator Lynx2. Russ J Bioorg Chem 46, 1261–1269 (2020). https://doi.org/10.1134/S1068162020060230

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