title: Enhanced in vitro refolding of soluble human glucocorticoid-induced TNF receptor-related ligand.
abstract: The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.
authors.affiliation: Department of Bioengineering, Sapientia Hungarian University of Transylvania, Piaţa Libertăţii 1, Miercurea Ciuc 530104, Romania.
authors.forename: Erika
authors.initials: E
authors.lastname: Kovács
authors.forename: László
authors.initials: L
authors.lastname: Szilágyi
authors.forename: Gábor
authors.initials: G
authors.lastname: Koncz
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Lányi
authors.forename: Beáta
authors.initials: B
authors.lastname: Ábrahám
mesh_terms: D001921:Brain / Q000166:cytology / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D053450:Glucocorticoid-Induced TNFR-Related Protein / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008024:Ligands; D010766:Phosphorylation; D017510:Protein Folding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D048069:Tumor Necrosis Factors / Q000235:genetics / Q000378:metabolism*; D020298:U937 Cells
chemical_list: D018076:DNA, Complementary; D053450:Glucocorticoid-Induced TNFR-Related Protein; D008024:Ligands; D011994:Recombinant Proteins; C491405:TNFRSF18 protein, human; C478967:TNFSF18 protein, human; D048069:Tumor Necrosis Factors