High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties
- Author(s)
- Hercus, TR; Barry, EF; DOTTORE, M; McClure, BJ; Webb, AI; Lopez, AF; Young, IG; Murphy, JM;
- Details
- Publication Year 2013-08-26,Volume 8,Issue #8,Page e74376
- Journal Title
- PLOS ONE
- Publication Type
- Journal Article
- 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 wildtype hIL-3, can be efficiently radio-iodinated for receptor binding studies.
- Publisher
- PUBLIC LIBRARY SCIENCE
- Keywords
- COLONY-STIMULATING FACTOR; RECOMBINANT HUMAN INTERLEUKIN-3; HEMATOPOIETIC STEM-CELLS; COMMON BETA-SUBUNIT; GM-CSF; RECEPTOR ACTIVATION; MURINE IL-3; EXPRESSION; BINDING; PURIFICATION
- Research Division(s)
- Cancer And Haematology; Structural Biology
- Publisher's Version
- https://doi.org/10.1371/journal.pone.0074376
- Open Access at Publisher's Site
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0074376- Terms of Use/Rights Notice
- Copyright: © 2013 Hercus et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Creation Date: 2013-08-26 12:00:00