Production, purification, and characterization of human recombinant IL-8 from the eucaryotic vector expression system baculovirus.
The cDNA for the human chemotactic interleukin, IL-8, was subcloned from a bacterial source into the eucaryotic vector expression system baculovirus. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from Sf9 cells derived from Spodoptera frugiperda following infection of a recombinant virus harboring the full-length IL-8 structural gene. Infected Sf9 cells produced rhIL-8 in a range from 0.5 to 2.0 mg of rhIL-8/liter of postinfection cell culture media. The recombinant interleukin was purified (greater than 600-fold) to homogeneity using preparative HPLC. rhIL-8 retained all of the physical, immunological, and biochemical properties observed for the natural product, monocyte-derived IL-8. rhIL-8 was assessed for biological efficacy by three criteria: (a) ability to induce chemotaxis in human neutrophils, (b) ability to induce oxygen burst metabolism, and (c) ability to be recognized by purified rabbit antibody generated against monocyte-derived IL-8. Antibody generated against monocyte-derived IL-8 recognized rhIL-8 isolated during all stages of the purification protocol. rhIL-8 was strongly chemotactic for human neutrophils and exhibited a chemotactic index comparable to that reported for other strong chemotactic peptides. rhIL-8 was identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single silver-stained band having an estimated molecular weight of 9.2 kDa and displayed amino acid residue molar abundance homology predicted for the mature form of the interleukin. Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production.
Kang
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XQ
Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107-2960.
Wiggins
J
J
Mallick
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Grant
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SR
D000596:Amino Acids / Q000032:analysis; D016367:Baculoviridae / Q000235:genetics; D002634:Chemotaxis, Leukocyte; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016897:Respiratory Burst
D000596:Amino Acids; D016209:Interleukin-8; D011994:Recombinant Proteins