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


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.
Autor Marta Szymczak (WCh / KChB / PChM)
Marta Szymczak
- Pracownia Chemii Medycznej
, Szymon Ziętkiewicz (MWB UG i GUMed / M020 / KBMiK)
Szymon Ziętkiewicz
- Katedra Biologii Molekularnej i Komórkowej
, Katarzyna Kuncewicz (WCh / KChB / PChM)
Katarzyna Kuncewicz
- Pracownia Chemii Medycznej
, Sylwia Rodziewicz-Motowidło (WCh / KChB / PChM)
Sylwia Rodziewicz-Motowidło
- Pracownia Chemii Medycznej
, Marta Orlikowska (WCh / KChB / PChM)
Marta Orlikowska
- Pracownia Chemii Medycznej
Tytuł czasopisma/seriiProtein Expression and Purification, ISSN 1046-5928, (N/A 70 pkt)
Rok wydania2019
Numer artykułu105450
Słowa kluczowe w języku angielskimHerpesvirus entry mediator, expression, purification, refolding
Klasyfikacja ASJC1305 Biotechnology
Języken angielski
Punktacja (całkowita)70
PunktacjaPunktacja MNiSW = 70.0, 30-09-2019, ArticleFromJournal
Wskaźniki publikacji Scopus SNIP (Source Normalised Impact per Paper): 2016 = 0.627; Impact Factor WoS: 2017 = 1.338 (2) - 2017=1.406 (5)
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