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Related Experiment Video

Updated: Mar 8, 2026

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
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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
PubMed
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.

Keywords:
LRP1 protein, humancoagulation factor VIIIhemophilia Alentivirusvon Willebrand factor

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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.