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Expression and characterization of a codonoptimized blood coagulation factor VIII

Shestopal, SA, Hao, J-J, Karnaukhova, E, Liang, Y, Ovanesov, MV, Lin, M, Kurasawa, JH, Lee, TK, McVey, John and Sarafanov, AG (2017) Expression and characterization of a codonoptimized blood coagulation factor VIII Journal of Thrombosis and Haemostasis, 15 (4). pp. 709-720.

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Abstract

Background: Production of recombinant factor VIII (FVIII) is challenging due to 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 expressed from codon-optimized (CO) and the wild-type (WT) cDNAs. Methods: Each variant of the BDD-FVIII was expressed in several independent 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 (including thrombin cleavage), mass-spectrometry, circular dichroism, surface plasmon resonance, chromogenic, clotting and thrombin generation assays. Results and Conclusion: The average yield of the CO was 7-fold higher than WT, while 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 averagely 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 due to 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.

Item Type: Article
Subjects : Biosciences and Medicine
Divisions : Faculty of Health and Medical Sciences > School of Biosciences and Medicine
Authors :
NameEmailORCID
Shestopal, SAUNSPECIFIEDUNSPECIFIED
Hao, J-JUNSPECIFIEDUNSPECIFIED
Karnaukhova, EUNSPECIFIEDUNSPECIFIED
Liang, YUNSPECIFIEDUNSPECIFIED
Ovanesov, MVUNSPECIFIEDUNSPECIFIED
Lin, MUNSPECIFIEDUNSPECIFIED
Kurasawa, JHUNSPECIFIEDUNSPECIFIED
Lee, TKUNSPECIFIEDUNSPECIFIED
McVey, Johnj.mcvey@surrey.ac.ukUNSPECIFIED
Sarafanov, AGUNSPECIFIEDUNSPECIFIED
Date : 21 February 2017
Identification Number : 10.1111/jth.13632
Copyright Disclaimer : This is the peer reviewed version of the following article: Shestopal SA, Hao J-J, Karnaukhova E, Liang Y, Ovanesov MV, Lin M, Kurasawa JH, Lee TK, McVey JH, Sarafanov AG. Expression and characterization of a codon-optimized blood coagulation factor VIII. J Thromb Haemost 2017; 15: 709–20, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/jth.13632/full. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving
Uncontrolled Keywords : Coagulation Factor VIII; LRP1 protein, human; von Willebrand Factor, Lentivirus; Hemophilia A
Related URLs :
Depositing User : Symplectic Elements
Date Deposited : 13 Jan 2017 14:54
Last Modified : 07 Jul 2017 10:59
URI: http://epubs.surrey.ac.uk/id/eprint/813284

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