Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin
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Thorough analysis of multimer composition and molecular structure of recombinant von Willebrand factor (r‐vWF) produced by recombinant CHO cells demonstrated r‐vWF to be more intact and less proteolytically degraded than plasma‐derived vWF (pd‐vWF) [B. Fischer et al. (1994) FEBS Lett. 351, 345–348]. In contrast to pd‐vWF, r‐vWF preparations consisted of pro‐vWF (vWF containing covalently attached propeptide) as well as mature vWF subunits forming homo‐ and hetero‐multimers. In order to ensure complete propeptide processing, a r‐vWF‐producing CHO cell clone was transfected with the cDNA of the human propeptide processing enzyme Furin. A r‐vWF/r‐Furin co‐expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters. r‐vWF obtained from these cells was fully processed. Analysis of r‐vWF by multimer analysis revealed a multimer pattern equal in number of high molecular weight multimer to pd‐vWF, but absence of satellite bands. Two‐dimensional electrophoretic analysis of both the primary dimer and the complete multimer pattern of r‐vWF showed that the recombinant coagulation factor was composed exclusively of intact and mature subunits. Since the triplet structure typical to pd‐vWF is known to reflect proteolytic degradation, r‐vWF thus exhibits an integrity far superior compared to pd‐vWF.
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Co-expression of furin allowed the production of fully cleaved and active von Willebrand factor in an industrial-scale CHO perfusion system.
“For example, co-expression of furin was shown to allow the production of fully cleaved and active von Willebrand factor in an industrial-scale CHO perfusion system (Fischer et al., 1995)”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Fischer et al., 1995
The Expression section describes furin-coexpressing CHO cells grown in industrial-scale perfusion bioreactors. The structural analysis (section labeled Preparation; page unavailable) reports that the resulting vWF was completely processed and composed of intact, mature subunits. It identifies multimers associated with hemostatic activity but does not demonstrate the produced vWF’s activity with a functional test. (LLM, conf=0.85)
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Therapeutic glycoprotein production in mammalian cells
Lalonde, M.-E., & Durocher, Y. (2017) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2017.04.028 · Cited by 296 · see citing works
- Authors
- Fischer, B. E., Schlokat, U., Mitterer, A., Reiter, M., Mundt, W., Turecek, P. L., Schwarz, H. P., & Dorner, F.
- Corresponding author
- Fischer, Bernhard E.
- Venue
- FEBS Letters
- Publisher
- Wiley
- Published
- 1995-11-20
- Volume
- 375
- Issue
- 3
- Pages
- 259-262
- ISSN
- 0014-5793 (ISSN-L)
- 1873-3468
- OpenAlex
- W1998279685
- Cited by
- 32
- References
- 0
- Citations by year
- 2026: 1 · 2019: 1 · 2017: 2 · 2016: 1
- FWCI
- 1.36
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- FEBS Letters (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Health Sciences › Medicine › Hematology › Platelet Disorders and Treatments
- Health Sciences › Medicine › Hematology › Hemophilia Treatment and Research
- Keywords
- von Willebrand factor, recombinant von Willebrand factor, furin, propeptide processing, proteolysis, CHO cell expression, recombinant coagulation factors, satellite bands
- MeSH terms
- Animals, Clone Cells, Electrophoresis, Agar Gel, Electrophoresis, Polyacrylamide Gel, Fermentation, Cricetinae, Humans, Protein Processing, Post-Translational, Recombinant Proteins, Subtilisins, Transfection, von Willebrand Factor, Immunoblotting, Electrophoresis, Gel, Two-Dimensional, Gene Expression, CHO Cells, DNA, Complementary, Furin
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