Antitumor activity of interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli.
Interleukin-21 (IL-21) has recently been identified as a novel 4-helix-bundle type I cytokine possessing a cytokine receptor gamma chain essential for the immune response. We report the preparation and functional characterization of Escherichia coli-expressed recombinant human IL-21 (rIL-21). The rIL-21, expressed as insoluble inclusion bodies in E. coli, was solubilized and then refolded by using a modified dialysis method. The introduction of redox reagents during refolding led to a dramatic increase in the refolding efficiency. Circular dichroism spectrum analysis showed that the refolded rIL-21 had an alpha-helix as a secondary structure, which is a characteristic of type I cytokines. Flow cytometry confirmed previous reports that rIL-21 binds to CD3-activated T cells (T-LAK) and to cell lines Raji, HL60, and Jurkat. rIL-21 stimulated the proliferation of T-LAK but not peripheral blood mononuclear cells, and this effect seems to be identical to that of co-stimulation with anti-CD3 antibody. Growth inhibition assay indicated that enhancement of the cytotoxicity of T-LAK to the human bile duct carcinoma TFK-1 depended on the concentration of rIL-21. Thus, refolded rIL-21 had activity identical to that of authentic IL-21 and enhanced the anti-tumor activity of T-LAK. These conclusions suggest the potential use of the refolded cytokine in adoptive immunotherapy using T-LAK cells and in the discovery of other functions of the cytokine.
Asano
Ryutaro
R
Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan.
Kudo
Toshio
T
Makabe
Koki
K
Tsumoto
Kouhei
K
Kumagai
Izumi
I
D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001483:Base Sequence; D003602:Cytotoxicity, Immunologic; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006801:Humans; D016219:Immunotherapy, Adoptive; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000737:chemistry; D007378:Interleukins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000276:immunology / Q000628:therapy; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D016176:T-Lymphocyte Subsets / Q000276:immunology; D014407:Tumor Cells, Cultured; D000098546:Interleukin-21
D000970:Antineoplastic Agents; D004274:DNA, Recombinant; D007378:Interleukins; D011994:Recombinant Proteins; D000098546:Interleukin-21