High yield production of a soluble human interleukin-3 variant from E. coli with wild-type bioactivity and improved radiolabeling properties
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Date
2013
Authors
Hercus, T.
Barry, E.
Dottore, M.
McClure, B.
Webb, A.
Lopez, A.
Young, I.
Murphy, J.
Editors
van Raaij, M.
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Journal article
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PLoS ONE, 2013; 8(8):e74376-e74376-8
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Timothy R. Hercus, Emma F. Barry, Mara Dottore, Barbara J. McClure, Andrew I. Webb, Angel F. Lopez, Ian G. Young, James M. Murphy
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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.
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© 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.