High yield production of a soluble human interleukin-3 variant from E. coli with wild-type bioactivity and improved radiolabeling properties

• Published in: PloS one Vol. 8; no. 8; p. e74376
• Main Authors: Hercus, Timothy R, Barry, Emma F, Dottore, Mara, McClure, Barbara J, Webb, Andrew I, Lopez, Angel F, Young, Ian G, Murphy, James M
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 26-08-2013; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Binding; Biological activity; Biology; Blood cells; Cancer; Cell culture; Cell survival; Chromatography, Reverse-Phase; Cloning; Cytokines; DNA Primers; E coli; Escherichia coli - genetics; Hematology; Hemopoiesis; Humans; Interleukin 3; Interleukin-3 - biosynthesis; Interleukin-3 - chemistry; Interleukin-3 - genetics; Interleukin-3 - physiology; Iodination; Leukemia; Mass Spectrometry; Medical research; Mice; Microorganisms; Molecular Sequence Data; Pathology; Protein folding; Proteins; Sequence Homology, Amino Acid; Shaking; Solubility; Substrates; Tyrosine; Australia; Victoria Australia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0074376

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.