Expression and characterization of a codon‐optimized blood coagulation factor VIII

• Published in: Journal of thrombosis and haemostasis Vol. 15; no. 4; pp. 709 - 720
• Main Authors: Shestopal, S. A., Hao, J.‐J., Karnaukhova, E., Liang, Y., Ovanesov, M. V., Lin, M., Kurasawa, J. H., Lee, T. K., Mcvey, J. H., Sarafanov, A. G.
• Format: Journal Article
• Language: English
• Published: England Elsevier Limited 01.04.2017
• Subjects: Animals; Cell culture; Cell Line; CHO Cells; coagulation factor VIII; Codon; Cricetinae; Cricetulus; DNA, Complementary - metabolism; Factor VIII - genetics; Factor VIII - metabolism; Genetic Vectors; Glycosylation; hemophilia A; Humans; Lentivirus; LRP1 protein, human; Mutation; Peptide Fragments - genetics; Protein Engineering; Protein Structure, Secondary; Proteins; Rodents; Structure-Activity Relationship; Tyrosine - chemistry; von Willebrand factor; coagulation factor VIII; hemophilia A; von Willebrand factor; LRP1 protein, human; lentivirus
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/jth.13632

Essentials Recombinant factor VIII (FVIII) is known to be expressed at a low level in cell culture. To increase expression, we used codon‐optimization of a B‐domain deleted FVIII (BDD‐FVIII). This resulted in 7‐fold increase of the expression level in cell culture. The biochemical properties of codon‐optimized BDD‐FVIII were similar to the wild‐type protein. Summary Background Production of recombinant factor VIII (FVIII) is challenging because of its low expression. It was previously shown that codon‐optimization of a B‐domain‐deleted FVIII (BDD‐FVIII) cDNA resulted in increased protein expression. However, it is well recognized that synonymous mutations may affect the protein structure and function. Objectives To compare biochemical properties of a BDD‐FVIII variants expressed from codon‐optimized and wild‐type cDNAs (CO and WT, respectively). Methods Each variant of the BDD‐FVIII was expressed in several independent Chinese hamster ovary (CHO) cell lines, generated using a lentiviral platform. The proteins were purified by two‐step affinity chromatography and analyzed in parallel by PAGE‐western blot, mass spectrometry, circular dichroism, surface plasmon resonance, and chromogenic, clotting and thrombin generation assays. Results and conclusion The average yield of the CO was 7‐fold higher than WT, whereas both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low‐density lipoprotein receptor‐related protein 1. The CO preparations had on average 1.5‐fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure as a result of considerably higher protein concentrations during the production. We concluded that the codon‐optimization of the BDD‐FVIII resulted in significant increase of its expression and did not affect the structure‐function properties.