title: Expression and characterization of a codon-optimized blood coagulation factor VIII.
abstract: 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.
  
  
  
  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.
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Shestopal
authors.affiliation: Poochon Scientific, Frederick, MD, USA.
authors.forename: J-J
authors.initials: JJ
authors.lastname: Hao
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: E
authors.initials: E
authors.lastname: Karnaukhova
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: M V
authors.initials: MV
authors.lastname: Ovanesov
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: M
authors.initials: M
authors.lastname: Lin
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Kurasawa
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Lee
authors.affiliation: School of Biosciences and Medicine, University of Surrey, Surrey, UK.
authors.forename: J H
authors.initials: JH
authors.lastname: Mcvey
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: A G
authors.initials: AG
authors.lastname: Sarafanov
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003062:Codon; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000378:metabolism; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D016086:Lentivirus; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry
chemical_list: C451034:B-domain-deleted factor VIII; D003062:Codon; D018076:DNA, Complementary; D010446:Peptide Fragments; C427184:recombinant factor VIII SQ; D014443:Tyrosine; D005169:Factor VIII
keywords: LRP1 protein, human; coagulation factor VIII; hemophilia A; lentivirus; von Willebrand factor