title: A single-step purification of biologically active recombinant human interleukin-5 from a baculovirus expression system.
abstract: Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant baculovirus-infected insect cells expressed high levels of rhIL-5 (5-15 mg/liter culture) of which > 90% was processed to the mature form and secreted into the culture medium. After removing cells by centrifugation, rhIL-5 was purified by first adjusting the culture medium to the calculated pI value of mature IL-5 (pI 7.44) and then passing the conditioned medium through tandem linked anion- and cation-exchange columns. The resulting pass-through fraction contained the rhIL-5 and was devoid of contaminating proteins. An optional hydrophobic-interaction chromatography step effectively concentrated the pure homodimeric N-glycosylated rhIL-5 with a high overall yield (> 90%). N-terminal amino acid sequence determination indicated that cleavage of the human IL-5 leader sequence in insect cells occurred between Ala19 and Ile20. Recombinant human IL-5 prepared by this procedure bound to the high-affinity IL-5 receptor present on an eosinophilic leukemia cell line and elicited a proliferative response in the IL-5-dependent murine B-cell line BCL1. This rapid and simple procedure for the expression and purification of mature rhIL-5 should therefore enable studies requiring large amounts of this cytokine.
authors.affiliation: Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
authors.forename: P M
authors.initials: PM
authors.lastname: Brown
authors.forename: M P
authors.initials: MP
authors.lastname: Scheid
authors.forename: G P
authors.initials: GP
authors.lastname: O'Neill
authors.forename: P C
authors.initials: PC
authors.lastname: Tagari
authors.forename: D W
authors.initials: DW
authors.lastname: Nicholson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D006801:Humans; D015848:Interleukin-5 / Q000235:genetics* / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D018123:Receptors, Interleukin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera
chemical_list: D018076:DNA, Complementary; D015848:Interleukin-5; D018123:Receptors, Interleukin; D011994:Recombinant Proteins