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Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2001 Aug; Vol. 22 (3), pp. 414-21. - Publication Year :
- 2001
-
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.<br /> (Copyright 2001 Academic Press.)
- Subjects :
- Animals
Baculoviridae genetics
Complement C1 Inactivator Proteins isolation & purification
Complement C1 Inactivator Proteins metabolism
Genetic Vectors
Humans
Polysaccharides chemistry
Recombinant Proteins biosynthesis
Recombinant Proteins isolation & purification
Spodoptera
Complement C1 Inactivator Proteins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1046-5928
- Volume :
- 22
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 11483003
- Full Text :
- https://doi.org/10.1006/prep.2001.1461