A convenient method for preparation of an engineered mouse interleukin-3 analog with high solubility and wild-type bioactivity

Abstract

Mouse interleukin-3 (mIL-3) is a critical cytokine regulator of myeloid cell differentiation, survival and activation, and consequently this cytokine has become a key reagent for hematological studies in the laboratory. Although bacterial expression has been used for the preparation of recombinant mIL-3 for more than 20 years, the resultant cytokine is known to exhibit poor solubility, be prone to aggregation, and may contain mispaired disulfide bonds. As a result, little structural characterization of mIL-3 has been possible to date. In the present work, we describe a convenient, inexpensive, and scalable protocol for preparing an mIL-3 analog with wild-type bioactivity from Escherichia coli via a simple purification scheme. This analog is typically expressed at >1 mg/l of shaking Super broth culture and, owing to solubility >5 mg/ml, structural studies in solution by nuclear magnetic resonance spectroscopy are feasible for mIL-3 for the first time.

Two Modes of β-Receptor Recognition Are Mediated by Distinct Epitopes on Mouse and Human Interleukin-3

“…From alignment of the hIL-3 and mIL-3 sequences, we identified the homologous, candidate receptorbinding residues in mIL-3 and prepared a panel of mIL-3 mutants in which the highly conserved residue, Glu 23 , and neighboring residues in the A-and C-helices were individually mutated to alanine. The A-helix glutamic acid residue is very highly conserved throughout IL-3 orthologs (21), and the homologous A-helix glutamic acid residues in the related cytokines, hGM-CSF, hIL-5, and hIL-3, are known to be to central to h␤c recognition (22)(23)(24)(25). Importantly, we found that the mIL-3 Glu 23 and the surrounding cluster comprising Lys 27 (A-helix), Glu 65 (adjacent to C-helix), Val 69 , Asn 73 , and Lys…”

“…A more comprehensive alignment of IL-3 ortholog sequences is published in Ref. 21. D, structural alignment of mIL-3, mGM-CSF, and mIL-5 amino acid sequences, with the highly conserved A-helix glutamic acid residue highlighted.…”

1H, 13C and 15N resonance assignments of a highly-soluble murine interleukin-3 analogue with wild-type bioactivity

“…The expression and purification of mIL-3 33-156 have been described elsewhere (Murphy et al 2009b). For NMR studies, uniform isotopic labelling of mIL-3 33-156 with 15 N or 15 N and 13 C was achieved by expressing proteins in E. coli BL21(DE3) cells cultured in M9 minimal medium containing 1 g/L of 15 N-NH 4 Cl as the sole nitrogen source Fig.…”

“…In vivo, the first 26 residues of the primary sequence are cleaved during secretion from IL-3 producing cells, such as mast cells and activated T cells, to yield a mature, bioactive protein comprising residues 27-166 of the primary transcript. Our prior work has established that truncation of residues 27-32 and 157-166 vastly improved the solution behaviour of mIL-3, with this engineered construct of mIL-3 33-156 retaining bioactivity equivalent to that of an E. coli-derived, commerciallysourced mIL-3 standard (Murphy et al 2009b). The chemical shift assignments reported here form the basis for characterizing the interaction between mIL-3 33-156 and b IL-3 in solution using NMR spectroscopy, which will be particularly valuable for defining the intrinsic determinants that permit mIL-3, but not IL-5 or GM-CSF, to bind b IL-3 directly with remarkable specificity.…”

“…Intriguingly, although IL-3, IL-5, or GM-CSF do not detectably bind bc-subunit in the absence of cytokine-specific a-subunits, mice possess an additional b-receptor, known as b IL-3 , that binds specifically and directly to mIL-3 with an affinity of *20 nM (Murphy and Young 2006). The mIL-3 construct used in this study comprises residues 33-156, mIL-3 33-156 (SWISS-PROT accession number: P01586) with the substitution of an alanine for Cys105 to eliminate a second disulfide bond (Cys105-Cys166) that is poorly conserved in orthologue sequences and is dispensable for mIL-3 bioactivity (Murphy et al 2009b). In vivo, the first 26 residues of the primary sequence are cleaved during secretion from IL-3 producing cells, such as mast cells and activated T cells, to yield a mature, bioactive protein comprising residues 27-166 of the primary transcript.…”