TY - JOUR
T1 - High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties
AU - Hercus, Timothy R.
AU - Barry, Emma F.
AU - Dottore, Mara
AU - McClure, Barbara J.
AU - Webb, Andrew I.
AU - Lopez, Angel F.
AU - Young, Ian G.
AU - Murphy, James M.
PY - 2013/8/26
Y1 - 2013/8/26
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84883140800&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0074376
DO - 10.1371/journal.pone.0074376
M3 - Article
SN - 1932-6203
VL - 8
JO - PLoS ONE
JF - PLoS ONE
IS - 8
M1 - e74376
ER -