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

Autor: Shestopal SA; Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA., Hao JJ; Poochon Scientific, Frederick, MD, USA., Karnaukhova E; Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA., Liang Y; Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA., Ovanesov MV; Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA., Lin M; Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA., Kurasawa JH; Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA., Lee TK; Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA., Mcvey JH; School of Biosciences and Medicine, University of Surrey, Surrey, UK., Sarafanov AG; Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
Jazyk: angličtina
Zdroj: Journal of thrombosis and haemostasis : JTH [J Thromb Haemost] 2017 Apr; Vol. 15 (4), pp. 709-720. Date of Electronic Publication: 2017 Feb 21.
DOI: 10.1111/jth.13632
Abstrakt: 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.
(© 2017 International Society on Thrombosis and Haemostasis.)
Databáze: MEDLINE
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