Antitumor activity of interleukin‐21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli

FEBS Letters

Kumagai, Izumi²  Asano, Ryutaro¹  Makabe, Koki²  Kudo, Toshio¹  Tsumoto, Kouhei² 

[1]Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan

[2]Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Aoba-ku, Sendai 980-8579, Japan

关键词: Interleukin-21;    Refolding;    Inclusion body;    Adoptive immunotherapy;    LAK;    IL-21;    interleukin-21;    rIL-21;    recombinant interleukin 21;    Fv;    antibody variable domain fragment;    SDS–PAGE;    sodium dodecyl sulfate–polyacrylamide gel electrophoresis;    PBS;    phosphate-buffered saline;    T-LAK;    lymphokine-activated killer with T cell phenotype;    PBMC;    peripheral blood mononuclear cell;    PCR;    polymerase chain reaction;    MTS;    3-(4;    5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt;    E:T;    effector:target ratio;    BrdU;    bromodeoxyuridine;    FITC;    fluorescein isothiocyanate;    ELISA;    enzyme-linked immunosorbent assay;    Ab;    antibody;    mAb;    monoclonal antibody;   

DOI  :  10.1016/S0014-5793(02)03254-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Interleukin-21 (IL-21) has recently been identified as a novel 4-helix-bundle type I cytokine possessing a cytokine receptor γ 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 α-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.

【 授权许可】

Unknown