Improved expression of recombinant human factor IX by co-expression of GGCX, VKOR and furin
Open accessAbstract
Recombinant human FIX concentrates (rhFIX) are essential in the treatment and prevention of bleeding in the bleeding disorder haemophilia B. However, due to the complex nature of FIX production yields are low which leads to high treatment costs. Here we report the production of rhFIX with substantially higher yield by co-expressing human FIX with GGCX (γ-glutamyl carboxylase), VKOR (vitamin K epoxide reductase) and furin (paired basic amino acid cleaving enzyme) in Chinese hamster ovary (CHO) cells. Our results show that controlled co-expression of GGCX with FIX is critical to obtain high rhFIX titre, and, that co-expression of VKOR further increased the yield of active rhFIX. Furin co-expression improved processing of the leader peptide of rhFIX but had a minor effect on yield of active rhFIX. The optimal expression level of GGCX was surprisingly low and required unusual engineering of expression vector elements. For VKOR and furin the control of expression was less critical and could be achieved by standard vector element. Using our expression vectors an rhFIX-producing clone with an expression level of up to 30 mg/L of active rhFIX was obtained. In addition an efficient single step purification method was developed to obtain pure and active rhFIX with up to 94 % yield.
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CHO cells have limited ability to gamma-carboxylate recombinant proteins such as clotting factors.
“CHO cells also have limited ability to gamma-carboxylate recombinant proteins such as clotting factors (Kumar 2015), even though some improvements have been achieved through metabolic engineering work (Rehemtulla et al., 1993;Liu et al., 2014).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Liu et al., 2014
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Research works printed but not linked: 20
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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
- Liu, J., Jonebring, A., Hagström, J., Nyström, A.-C., & Lövgren, A.
- Affiliations
- AstraZeneca (Sweden) (SE)
- Corresponding author
- Lövgren, Ann
- ORCID
- Liu, Jianming
- Venue
- The Protein Journal
- Published
- 2014-02-25
- Volume
- 33
- Issue
- 2
- Pages
- 174-183
- ISSN
- 1572-3887 (ISSN-L)
- 1875-8355
- PMC
- PMC3947961
- OpenAlex
- W2076571925
- Cited by
- 20
- References
- 0
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2022: 2 · 2021: 1 · 2019: 3 · 2018: 1 · 2017: 3 · 2016: 6 · 2015: 1
- FWCI
- 2.36
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- The Protein Journal (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- AstraZeneca
- Topics
- Health Sciences › Medicine › Hematology › Hemophilia Treatment and Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- hemophilia B, gamma-glutamyl carboxylase, vitamin K epoxide reductase, furin, co-expression, CHO cells, protein yield, one-step purification
- MeSH terms
- Amino Acid Sequence, Animals, Cricetulus, Factor IX, Genetic Vectors, Cricetinae, Humans, Molecular Sequence Data, Recombinant Proteins, Gene Expression, CHO Cells, Carbon-Carbon Ligases, Furin, Vitamin K Epoxide Reductases
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