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

doi:10.1016/j.pep.2019.105450

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

Protein Expr Purif. 2019 Dec:164:105450. doi: 10.1016/j.pep.2019.105450. Epub 2019 Jul 9.

Authors

Marta Szymczak¹, Szymon Ziętkiewicz², Katarzyna Kuncewicz¹, Sylwia Rodziewicz-Motowidło¹, Marta Orlikowska³

Affiliations

¹ Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, 80-308, Gdansk, Wita Stwosza 63, Poland.
² Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk, 80-822, Gdansk, Kladki 24, Poland.
³ Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, 80-308, Gdansk, Wita Stwosza 63, Poland. Electronic address: marta.orlikowska@ug.edu.pl.

PMID: 31299214
DOI: 10.1016/j.pep.2019.105450

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 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.

Keywords: Expression; Herpesvirus entry mediator; Purification; Refolding.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Escherichia coli / genetics*
Gene Expression
Humans
Protein Domains
Protein Refolding*
Receptors, Tumor Necrosis Factor, Member 14 / chemistry*
Receptors, Tumor Necrosis Factor, Member 14 / genetics*
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Solubility

Substances

Receptors, Tumor Necrosis Factor, Member 14
Recombinant Proteins