Expression, purification, and efficient refolding of the extracellular domain of Escherichia coli - expressed signaling receptor herpesvirus entry mediator

Marta Szymczak , Szymon Ziętkiewicz , Katarzyna Kuncewicz , Sylwia Rodziewicz-Motowidło , Marta Orlikowska

Abstract

Herpesvirus entry mediator (HVEM), a member of the TNF-receptor superfamily, plays an important role in the regulation of the immune system. It forms a complex with ligands and can either activate or inhibit the response of the immune system. Furthermore, HVEM can exhibit pro-inflammatory or anti-inflammatory effects in many human diseases. Therefore, understanding the mechanism underlying the interaction of HVEM with other receptors is extremely important to design small therapeutic molecules that can stimulate the response of the immune system. In this study, we attempted to develop the most efficient method for the expression and purification of the extracellular domain of HVEM using Escherichia coli. The soluble fraction constituted only a small portion of the E. coli-expressed protein, whereas most majority of the protein was found to be accumulated in the insoluble fraction. Three different protein refolding methods were analyzed: dialysis, dilution, and using chromatographic column. The oligomeric state of the protein was determined by characterizing the obtained fractions using analytical size exclusion chromatography. All the obtained fractions were tested for their ability to form a complex with B- and T-lymphocyte attenuator using enzyme-linked immunosorbent assay. The results of this study provide crucial information regarding the production of HVEM protein in a robust, well-established, and convenient heterologous expression system using E. coli as a host. In addition, it allows for the selection of the most effective method for appropriate refolding of HVEM protein, which gets accumulated in the insoluble fraction.
Publication typeIn press (online first, early view)
Author Marta Szymczak (FCh / DBCh / LMCh)
Marta Szymczak,,
- Laboratory of Medical Chemistry
, Szymon Ziętkiewicz (IFB / M020 / DMCB)
Szymon Ziętkiewicz,,
- Department of Molecular and Cellular Biology
, Katarzyna Kuncewicz (FCh / DBCh / LMCh)
Katarzyna Kuncewicz,,
- Laboratory of Medical Chemistry
, Sylwia Rodziewicz-Motowidło (FCh / DBCh / LMCh)
Sylwia Rodziewicz-Motowidło,,
- Laboratory of Medical Chemistry
, Marta Orlikowska (FCh / DBCh / LMCh)
Marta Orlikowska,,
- Laboratory of Medical Chemistry
Journal seriesProtein Expression and Purification, ISSN 1046-5928, (A 15 pkt)
Issue year2019
Noonline first
Pages1-26
Publication size in sheets1.25
Article number105450
Keywords in EnglishHerpesvirus entry mediator, expression, purification, refolding
ASJC Classification1305 Biotechnology
DOIDOI:10.1016/j.pep.2019.105450
URL https://doi.org/10.1016/j.pep.2019.105450
Languageen angielski
Score (nominal)15
ScoreMinisterial score = 15.0, 18-07-2019, ArticleFromJournal
Ministerial score (2013-2016) = 15.0, 18-07-2019, ArticleFromJournal
Publication indicators Scopus SNIP (Source Normalised Impact per Paper): 2016 = 0.627; WoS Impact Factor: 2017 = 1.338 (2) - 2017=1.406 (5)
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