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
AbstractHerpesvirus 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.
|Journal series||Protein Expression and Purification, ISSN 1046-5928, (A 15 pkt)|
|Keywords in English||Herpesvirus entry mediator, expression, purification, refolding|
|Score||= 15.0, 02-09-2019, ArticleFromJournal|
|Publication indicators||: 2016 = 0.627; : 2017 = 1.338 (2) - 2017=1.406 (5)|
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