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Bacterial expression of the water-soluble domain of lynx1, an endogenous neuromodulator of human nicotinic receptors

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Abstract

The membrane-associated protein Lynx1 is expressed in the human central nervous system and plays an important role in the regulation of the activity of nicotinic acetylcholine receptors. In the present study, ws-Lynx1 encoding the water-soluble domain of human Lynx1 was cloned and studied by heterological expression in E. coli. In milligram quantities, the ws-Lynx1 protein could only be obtained in cytoplasmic inclusion bodies of bacterial cells. To optimize the yield of ws-Lynx1 recombinant protein, a system was developed that allowed retrieving functionally active ws-Lynx1 from the inclusion bodies. After renaturation, the protein was characterized by mass spectrometry and by circular dichroism spectroscopy. ws-Lynx1 was shown to inhibit the binding of [125I]-α-bungarotoxin to membranes from the electric organ of the Torpedo californica ray containing muscle-type nicotinic acetylcholine receptors (α12βγδ) in a competitive manner.

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Abbreviations

DTT:

dithiothreitol

GSH and GSSG:

reduced and oxidized glutathione

GST:

glutathione-S-transferase

GPI:

glycosylphosphatidylinositol

IC50:

inhibition constant corresponding to 50 percent blocking of receptor binding sites

nAChR:

nicotinic acetylcholine receptor

NT:

neurotoxin

STII:

signal peptide of E. coli enterotoxin II

TRX:

thioredoxin

ws-Lynx1:

water-soluble domain of the human neuromodulator Lynx1

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117991, Russia

    M. A. Shulepko, E. N. Lyukmanova, I. E. Kasheverov, D. A. Dolgikh, V. I. Tsetlin & M. P. Kirpichnikov

  2. Moscow State University, Moscow, 119991, Russia

    D. A. Dolgikh & M. P. Kirpichnikov

Authors
  1. M. A. Shulepko
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  2. E. N. Lyukmanova
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  3. I. E. Kasheverov
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  4. D. A. Dolgikh
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  5. V. I. Tsetlin
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  6. M. P. Kirpichnikov
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Corresponding author

Correspondence to E. N. Lyukmanova.

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Original Russian Text © M.A. Shulepko, E.N. Lyukmanova, I.E. Kasheverov, D.A. Dolgikh, V.I. Tsetlin, M.P. Kirpichnikov, 2011, published in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 5, pp. 609–615.

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Shulepko, M.A., Lyukmanova, E.N., Kasheverov, I.E. et al. Bacterial expression of the water-soluble domain of lynx1, an endogenous neuromodulator of human nicotinic receptors. Russ J Bioorg Chem 37, 543–549 (2011). https://doi.org/10.1134/S1068162011050165

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  • DOI: https://doi.org/10.1134/S1068162011050165

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