title: High yield production of a soluble human interleukin-3 variant from E. coli with wild-type bioactivity and improved radiolabeling properties.
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.
authors.affiliation: Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Hercus
authors.forename: Emma F
authors.initials: EF
authors.lastname: Barry
authors.forename: Mara
authors.initials: M
authors.lastname: Dottore
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: McClure
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Webb
authors.forename: Angel F
authors.initials: AF
authors.lastname: Lopez
authors.forename: Ian G
authors.initials: IG
authors.lastname: Young
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056148:Chromatography, Reverse-Phase; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007377:Interleukin-3 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012995:Solubility
chemical_list: D017931:DNA Primers; D007377:Interleukin-3