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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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Brown PM,Scheid MP,O'Neill GP,Tagari PC,Nicholson DW

doi

10.1006/prep.1995.1009

subject

Has Abstract

pub_date

1995-02-01 00:00:00

pages

63-71

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046592885710091

journal_volume

6

pub_type

杂志文章

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