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Protein Expr Purif. 2014 Oct;102:76-84. doi: 10.1016/j.pep.2014.07.002. Epub 2014 Aug 11.

Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag.

Author information

1
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
2
Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA.
3
Human Retrovirus Pathogenesis Section, Vaccine Branch-National Cancer Institute, Frederick, MD 21702-1201, USA.
4
Human Retrovirus Section, Vaccine Branch-National Cancer Institute, Frederick, MD 21702-1201, USA.
5
Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA. Electronic address: zaharoff@uark.edu.
6
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: sthalla@uark.edu.

Abstract

Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous. Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ∼98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichroism, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31±0.05 mg/mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app)=69±1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics.

KEYWORDS:

Bioactivity; Cytokine; HEK293; Heparin Sepharose; Interleukin-12; Purification

PMID:
25123642
PMCID:
PMC4164965
DOI:
10.1016/j.pep.2014.07.002
[Indexed for MEDLINE]
Free PMC Article

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