Abstract
Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in E. coli. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in E. coli as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL‑3, can be efficiently radio-iodinated for receptor binding studies.
Highlights
Human interleukin-3 is a four-helix bundle, short chain cytokine that is widely expressed in vivo, principally by hematopoietic cells, such as activated T-lymphocytes, mast cells and basophils [1]
Wild-type recombinant Human interleukin-3 (hIL-3) expressed in E. coli exhibits limited solubility [17] and has been typically produced by oxidative refolding of material expressed in inclusion bodies, even by commercial suppliers
Since the human IL-3 gene was cloned in 1986 [28], highyielding, cost-effective approaches have been sought for the expression and purification of hIL-3 for biochemical, structural and biological studies, including as a growth stimulus for cell culture applications
Summary
Human interleukin-3 (hIL-3) is a four-helix bundle, short chain cytokine that is widely expressed in vivo, principally by hematopoietic cells, such as activated T-lymphocytes, mast cells and basophils [1]. The effects of IL-3 on target cells are initiated by IL-3 binding to a transmembrane receptor system composed of an IL-3specific α-subunit (IL-3Rα) and a common β-subunit (βc) shared with the related cytokines, IL-5 and GM-CSF [14,15,16]. The method described provides a robust and convenient strategy to produce milligram quantities of fully bioactive soluble hIL‐3 for a broad range of laboratory applications, including hematopoietic cell culture and molecular characterization of hIL-3 receptor binding
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