A convenient method for preparation of an engineered mouse interleukin-3 analog with high solubility and wild-type bioactivity.
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.
Murphy
James M
JM
Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia. jamesm@wehi.edu.au
Metcalf
Donald
D
Young
Ian G
IG
Hilton
Douglas J
DJ
D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D007377:Interleukin-3 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D012995:Solubility
D016207:Cytokines; D007377:Interleukin-3