title: Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Jayanthi
authors.affiliation: Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA.
authors.forename: Bhanu prasanth
authors.initials: Bp
authors.lastname: Koppolu
authors.affiliation: Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA.
authors.forename: Sean G
authors.initials: SG
authors.lastname: Smith
authors.affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch-National Cancer Institute, Frederick, MD 21702-1201, USA.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Jalah
authors.affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch-National Cancer Institute, Frederick, MD 21702-1201, USA.
authors.forename: Jenifer
authors.initials: J
authors.lastname: Bear
authors.affiliation: Human Retrovirus Section, Vaccine Branch-National Cancer Institute, Frederick, MD 21702-1201, USA.
authors.forename: Margherita
authors.initials: M
authors.lastname: Rosati
authors.affiliation: Human Retrovirus Section, Vaccine Branch-National Cancer Institute, Frederick, MD 21702-1201, USA.
authors.forename: George N
authors.initials: GN
authors.lastname: Pavlakis
authors.affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch-National Cancer Institute, Frederick, MD 21702-1201, USA.
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Felber
authors.affiliation: Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA. Electronic address: zaharoff@uark.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Zaharoff
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: sthalla@uark.edu.
authors.forename: Thallapuranam Krishnaswamy Suresh
authors.initials: TK
authors.lastname: Kumar
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D018664:Interleukin-12 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D011994:Recombinant Proteins; D018664:Interleukin-12; D006493:Heparin
keywords: Bioactivity; Cytokine; HEK293; Heparin Sepharose; Interleukin-12; Purification