Expression, purification, and efficient refolding of the extracellular domain of Escherichia coli-expressed signaling receptor herpesvirus entry mediator.
Protein Expr Purif
; 164: 105450, 2019 12.
Article
en En
| MEDLINE
| ID: mdl-31299214
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_neglected_diseases
/
3_zoonosis
Asunto principal:
Escherichia coli
/
Miembro 14 de Receptores del Factor de Necrosis Tumoral
/
Replegamiento Proteico
Límite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2019
Tipo del documento:
Article
País de afiliación:
Polonia