Production of recombinant human monocyte/neutrophil elastase inhibitor (rM/NEI).
Recombinant human monocyte/neutrophil elastase inhibitor (rM/NEI) was expressed with a baculovirus expression system. The purified recombinant protein was shown to inhibit human neutrophil elastase by the formation of a stable equimolar complex, as had been shown for M/NEI isolated from monocyte-derived cell lines. rM/NEI was remarkably stable in aqueous buffers from pH 6 to pH 8, but not in buffers below pH 6. rM/NEI activity was stable when subjected to freeze-thaw cycles and low temperature storage in Tris or phosphate buffers. rM/NEI could also be lyophilized without significant loss of activity. A 1.6-g batch of greater than 95% purity in rM/NEI was obtained by anion exchange and size exclusion chromatography with yields of 7 to 8 mg per liter of cultured insect cells. Methods and protocols were chosen for compatibility with large-scale cGMP production and were suitable for biochemical characterization and preclinical evaluation of rM/NEI as a therapeutic agent for cystic fibrosis. The availability of large amounts of purified rM/NEI will facilitate clinical evaluation of rM/NEI for prevention of the elastase-mediated destruction of lung tissue associated with the morbidity and mortality of cystic fibrosis.
Center for Blood Research, Harvard Medical School and CBR Laboratories, Inc., Boston, Massachusetts, 02115, USA.
J
J
Cooley
B
B
Mathieu
E
E
Remold-O'Donnell
R J
RJ
Mandle
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D004355:Drug Stability; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors; D017924:Nucleopolyhedroviruses / Q000235:genetics; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015843:Serpins; D018411:Spodoptera
D000596:Amino Acids; D011506:Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C075177:SERPINB1 protein, human; D019272:Leukocyte Elastase