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Updated: Mar 8, 2026
12:24
Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
Published on: June 3, 2014
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Expression and characterization of a codon-optimized blood coagulation factor VIII.
S A Shestopal1, J-J Hao2, E Karnaukhova1
1Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
Journal of Thrombosis and Haemostasis : JTH
|January 22, 2017
Summary
Codon-optimization significantly increased recombinant factor VIII (FVIII) expression by 7-fold in cell culture without altering its biochemical properties or function compared to wild-type protein.
Area of Science:
- Biotechnology
- Protein Engineering
- Molecular Biology
Background:
- Recombinant factor VIII (FVIII) production is hindered by low expression levels in cell culture.
- Codon-optimization of B-domain deleted FVIII (BDD-FVIII) has shown potential for increased protein yield.
- Synonymous mutations from codon-optimization could potentially impact protein structure and function.
Purpose of the Study:
- To compare the biochemical properties of codon-optimized (CO) BDD-FVIII with wild-type (WT) BDD-FVIII.
- To assess the impact of codon-optimization on the structure-function relationship of recombinant FVIII.
Main Methods:
- Expressed CO and WT BDD-FVIII variants in Chinese hamster ovary (CHO) cell lines using a lentiviral platform.
- Purified proteins via two-step affinity chromatography.
- Analyzed proteins using PAGE-western blot, mass spectrometry, circular dichroism, surface plasmon resonance, and functional assays (chromogenic, clotting, thrombin generation).
Main Results:
- Codon-optimized BDD-FVIII yielded 7-fold higher protein expression compared to WT.
- Both CO and WT proteins exhibited highly similar amino acid sequences, fragmentation patterns, glycosylation, and binding affinities.
- CO preparations demonstrated a 1.5-fold increase in specific activity, attributed to better structural preservation during production.
Conclusions:
- Codon-optimization of BDD-FVIII significantly enhances protein expression levels.
- The codon-optimization strategy does not adversely affect the structural integrity or functional properties of BDD-FVIII.
- This approach offers a promising method for improving the production of recombinant FVIII.
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