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

J Thromb Haemost. 2017 Apr;15(4):709-720. doi: 10.1111/jth.13632. Epub 2017 Feb 21.

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.

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.

Keywords: LRP1 protein, human; coagulation factor VIII; hemophilia A; lentivirus; von Willebrand factor.

Publication types

  • Research Support, U.S. Gov't, P.H.S.
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • CHO Cells
  • Cell Line
  • Codon*
  • Cricetinae
  • Cricetulus
  • DNA, Complementary / metabolism
  • Factor VIII / genetics*
  • Factor VIII / metabolism
  • Genetic Vectors
  • Glycosylation
  • Humans
  • Lentivirus
  • Mutation
  • Peptide Fragments / genetics
  • Protein Engineering*
  • Protein Structure, Secondary
  • Structure-Activity Relationship
  • Tyrosine / chemistry

Substances

  • B-domain-deleted factor VIII
  • Codon
  • DNA, Complementary
  • Peptide Fragments
  • recombinant factor VIII SQ
  • Tyrosine
  • Factor VIII

Grant support