ArticleProtein expression and purification2014
Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag.
Article in Protein expression and purification, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 25 citations in OpenAlex.
- An injectable hydrogel enhances intratumoral retention and antitumor efficacy of cytokine immunotherapy in murine triple negative breast tumor models.Journal of controlled release : official journal of the Controlled Release Society · 2025Article
- Focal Cryo-Immunotherapy with Intratumoral IL-12 Prevents Recurrence of Large Murine Tumors.Cancers · 2023Article
- Heparin and Heparin-Based Drug Delivery Systems: Pleiotropic Molecular Effects at Multiple Drug Resistance of Osteosarcoma and Immune Cells.Pharmaceutics · 2022Review
- Intranasal Delivery of Thermostable Subunit Vaccine for Cross-Reactive Mucosal and Systemic Antibody Responses Against SARS-CoV-2.Frontiers in immunology · 2022Article
- Safety and Pharmacokinetics of Intravesical Chitosan/Interleukin-12 Immunotherapy in Murine Bladders.Bladder cancer (Amsterdam, Netherlands) · 2021Article
- Localized Interleukin-12 for Cancer Immunotherapy.Frontiers in immunology · 2020Review
- Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein.Cytokine · 2019Article
- Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE.Molecules (Basel, Switzerland) · 2019Article
- Molecular mechanisms of heparin-induced modulation of human interleukin 12 bioactivity.The Journal of biological chemistry · 2019Article
- The localisation of the heparin binding sites of human and murine interleukin-12 within the carboxyterminal domain of the P40 subunit.Cytokine · 2018Article
- Modulation of Interleukin-12 activity in the presence of heparin.Scientific reports · 2017Article
Corrections and comments
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Authors and funding
10 authors at 2 institutions in 1 country.
Funding
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.