High Yield Production of a Soluble Human Interleukin-3 Variant from <i>E. coli</i> with Wild-Type Bioactivity and Improved Radiolabeling Properties

<div><p>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 <i>E. coli</i>. 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 <i>E. coli</i> 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.</p> </div

Purification of the hIL-3(13-125; W13Y) analog.

<div><p>A) Workflow diagram illustrating the purification protocol for hIL-3(13-125; W13Y).</p> <p>B) Elution profile of the TEV protease digested, NusA and hIL-3 analog mixture, following size exclusion chromatography (SEC) using a Superdex 200 column (26 mm x 600 mm) operated at 2 ml/min at 4°C with 50 mM sodium phosphate pH 7.0, 150 mM NaCl as running buffer. Free NusA eluted at ~186 mL and the hIL-3 analog eluted at 254 mL. The first peak at 116 mL contains aggregates while we suspect the last peak at 328 mL contains DTT from the digest. Molecular weight (kDa) marker elution positions are marked as dots above the elution profile.</p> <p>C) Analysis of hIL-3 analog purification by 15% acrylamide reducing SDS-PAGE with Coomassie Blue staining. NusA: hIL-3(13-125; W13Y) fusion protein was isolated by Ni-chromatography (Lane 1) prior to cleavage by TEV protease (Lane 2) to yield free NusA (55kDa) and the hIL-3 analog (13.4kDa). Fractions containing the hIL-3 analog that eluted around 254ml during SEC are shown (Lanes 4-9), illustrating that the hIL-3 analog was purified free from NusA (Lane 3).</p> <p>D) The SEC purified hIL-3 analog was applied to an Aquapore RP300 reversed-phase column (4.6 mm x100 mm) and bound proteins eluted using a 0-100% gradient of acetonitrile in 0.1% trifluoroacetic acid. The hIL-3 analog eluted at 34 min (~50% acetonitrile).</p> <p>E) Purified hIL-3(13-125; W13Y) was subjected to tryptic digestion and tandem mass spectrometry. The sequences of peptides not identified in this analysis are shown as lowercase italics. Asterisked methionine residues were oxidized. Sequence arising from the NusA-His₆ fusion overhang after TEV protease cleavage is underlined, while the residues are numbered according to the mature, full-length IL-3 reference sequence.</p></div

Alignment of amino acid sequences of human and mouse IL-3.

<p>The amino acid sequences of full length wild-type hIL-3, hIL-3(13-125; W13Y), the hIL-3 analog SC-65369 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074376#B41" target="_blank">41</a>], and wild-type full length mouse IL-3 were manually aligned owing to low homology between mouse and human IL-3 (29% identity). Numbers above the sequence refer to the mature form of full length hIL-3 with dots above every 10^th residue. The sequences shown in gray for full length hIL-3 and full length mouse IL-3 are signal peptides that are cleaved during secretion. The key substitution, W13Y in hIL-3(13-125; W13Y), is shown in bold text and highlighted.</p