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On-column refolding and characterization of soluble human interleukin-15 receptor alpha-chain produced in Escherichia coli.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2003 Sep; Vol. 31 (1), pp. 64-71. - Publication Year :
- 2003
-
Abstract
- Interleukin-15 receptor alpha-chain (IL-15Ralpha) is a member of the new cytokine receptor family, which possesses the sushi domain. To investigate the biochemical and biophysical characteristics of soluble human IL-15Ralpha (shIL-15Ralpha), shIL-15Ralpha was recombinantly expressed in Escherichia coli. The shIL-15Ralpha containing a six histidine-tag was expressed as inclusion bodies, which were solubilized with urea, immobilized on a Ni-nitrilotriacetic acid column, and refolded by a decreasing gradient of urea concentration. The refolded shIL-15Ralpha exhibited a highly flexible structure, neutralized human interleukin-15-induced cell proliferation effectively, and bound to its ligand with the same affinity as human IL-15Ralpha on the cell surface, as demonstrated by circular dichroism, a cell proliferation assay, and surface plasmon resonance, respectively. Thus, we succeeded in refolding shIL-15Ralpha to an active form on an affinity column.
- Subjects :
- Amino Acid Sequence
Antibodies immunology
Binding, Competitive
Blotting, Western
Cell Division drug effects
Cell Line
Chromatography, Gel
Circular Dichroism
DNA, Complementary genetics
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Escherichia coli metabolism
Gene Expression
Genetic Vectors genetics
Histidine genetics
Humans
Interleukin-15 metabolism
Interleukin-15 pharmacology
Isoelectric Focusing
Kinetics
Mercaptoethanol chemistry
Molecular Sequence Data
Protein Binding
Protein Structure, Secondary
Receptors, Interleukin-15
Receptors, Interleukin-2 genetics
Receptors, Interleukin-2 metabolism
Recombinant Proteins genetics
Recombinant Proteins metabolism
Sequence Analysis, Protein
Spectrometry, Mass, Electrospray Ionization
Spleen chemistry
Surface Plasmon Resonance
T-Lymphocytes, Cytotoxic drug effects
T-Lymphocytes, Cytotoxic metabolism
Urea chemistry
Escherichia coli genetics
Protein Folding
Receptors, Interleukin-2 chemistry
Recombinant Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1046-5928
- Volume :
- 31
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 12963342
- Full Text :
- https://doi.org/10.1016/s1046-5928(03)00143-8