Abstract
Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.
Highlights
Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine important for the stimulation of proliferation, differentiation and survival of many hemopoietic cells [1] including mature neutrophils, macrophages and dendritic cells [2,3]
We describe in detail here a straightforward method to refold and purify rhGM-CSF from inclusion bodies that generates milligram amounts of active protein from a single litre of E. coli
Through prior variation of induction times and lengths, it was found that addition of 1 mM isopropyl b-D-1-thiogalatopyranoside (IPTG) at a culture optical density (600 nm) of approximately 0.3 and subsequent culturing for 5 hrs at 37uC yielded the highest expression of rhGM-CSF
Summary
Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine important for the stimulation of proliferation, differentiation and survival of many hemopoietic cells [1] including mature neutrophils, macrophages and dendritic cells [2,3]. GM-CSF is produced by many cell types within the body (e.g. fibroblasts, endothelial cells) when stimulated by microbial products or inflammatory cytokines and its activity is important to the innate immune response. Because of its ability to stimulate hemopoietic cells, recombinant human GMCSF (rhGM-CSF) is used, for instance, as a biotherapeutic for immunocompromised individuals undergoing chemotherapy [4]. Dysregulation of GM-CSF activity has been implicated in such auto-immune conditions as arthritis and multiple sclerosis [5]. Neutralization of the bioactivity of GM-CSF by auto-antibodies causes another auto-immune disease, idiopathic pulmonary alveolar proteinosis [6] and GM-CSF is used to treat this condition [7,8]
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