On-column refolding and characterization of soluble human interleukin-15 receptor alpha-chain produced in Escherichia coli.
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.
Matsumoto
Mitsuhiro
M
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, 3-17-35 Niizo-Minami, Toda, 335-8502, Saitama, Japan.
Misawa
Satoru
S
Tsumoto
Kouhei
K
Kumagai
Izumi
I
Hayashi
Hideya
H
Kobayashi
Yoshiro
Y
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001667:Binding, Competitive; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D019409:Interleukin-15 / Q000378:metabolism / Q000494:pharmacology; D007525:Isoelectric Focusing; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D053647:Receptors, Interleukin-15; D015375:Receptors, Interleukin-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D013154:Spleen / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D000906:Antibodies; D018076:DNA, Complementary; C508024:IL15RA protein, human; D019409:Interleukin-15; D053647:Receptors, Interleukin-15; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D008623:Mercaptoethanol; D014508:Urea