title: Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization.
abstract: C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Engineering, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Wolff
authors.forename: F
authors.initials: F
authors.lastname: Zhang
authors.forename: J J
authors.initials: JJ
authors.lastname: Roberg
authors.forename: E E
authors.initials: EE
authors.lastname: Caldwell
authors.forename: P R
authors.initials: PR
authors.lastname: Kaul
authors.forename: J N
authors.initials: JN
authors.lastname: Serrahn
authors.forename: D W
authors.initials: DW
authors.lastname: Murhammer
authors.forename: R J
authors.initials: RJ
authors.lastname: Linhardt
authors.forename: J M
authors.initials: JM
authors.lastname: Weiler
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003174:Complement C1 Inactivator Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D018411:Spodoptera
chemical_list: D003174:Complement C1 Inactivator Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins