Journal article
Expression of C1 Esterase Inhibitor by the Baculovirus Expression Vector System: Preparation, Purification, and Characterization
Protein expression and purification, Vol.22(3), pp.414-421
08/2001
DOI: 10.1006/prep.2001.1461
PMID: 11483003
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 μg 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.
Details
- Title: Subtitle
- Expression of C1 Esterase Inhibitor by the Baculovirus Expression Vector System: Preparation, Purification, and Characterization
- Creators
- Michael W Wolff - Department of Chemical and Biochemical Engineering, College of EngineeringFuming Zhang - Department of Chemical and Biochemical Engineering, College of EngineeringJeff J Roberg - Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City, Iowa, 52242Elizabeth E.O Caldwell - Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City, Iowa, 52242Patrick R Kaul - Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City, Iowa, 52242Jill N Serrahn - Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City, Iowa, 52242David W Murhammer - Department of Chemical and Biochemical Engineering, College of EngineeringRobert J Linhardt - Department of Chemical and Biochemical Engineering, College of EngineeringJohn M Weiler - Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City, Iowa, 52242
- Resource Type
- Journal article
- Publication Details
- Protein expression and purification, Vol.22(3), pp.414-421
- Publisher
- Elsevier Inc
- DOI
- 10.1006/prep.2001.1461
- PMID
- 11483003
- ISSN
- 1046-5928
- eISSN
- 1096-0279
- Language
- English
- Date published
- 08/2001
- Academic Unit
- Chemical and Biochemical Engineering; Internal Medicine
- Record Identifier
- 9984003475602771
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