Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
Poochon Scientific, Frederick, MD, USA.
J Thromb Haemost. 2017 Apr;15(4):709-720. doi: 10.1111/jth.13632. Epub 2017 Feb 21.
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.
已知因子 VIII（FVIII）在细胞培养中表达水平较低。为了提高表达水平，我们使用 B 结构域缺失的 FVIII（BDD-FVIII）的密码子优化。这导致细胞培养中表达水平提高了 7 倍。密码子优化的 BDD-FVIII 的生化特性与野生型蛋白相似。
背景因子 VIII（FVIII）的表达具有挑战性，因为其表达水平较低。先前的研究表明，B 结构域缺失的 FVIII（BDD-FVIII）cDNA 的密码子优化导致蛋白表达增加。然而，人们普遍认识到同义突变可能会影响蛋白的结构和功能。目的比较来自密码子优化和野生型 cDNA（分别为 CO 和 WT）表达的 BDD-FVIII 变体的生化特性。方法使用慢病毒平台，在几个独立的中国仓鼠卵巢（CHO）细胞系中表达每种 BDD-FVIII 变体。通过两步亲和层析纯化蛋白，并通过 PAGE-免疫印迹、质谱、圆二色性、表面等离子体共振和显色、凝固和凝血酶生成分析平行分析。结果和结论 CO 的平均产量比 WT 高 7 倍，而两种蛋白的氨基酸序列完全相同（99%覆盖率），并且在分子片段的模式（凝血酶切割前后）、糖基化和酪氨酸硫酸化、二级结构和与 von Willebrand 因子以及 LDL 受体相关蛋白 1 的片段的结合方面非常相似。CO 制剂的 FVIII 比 WT 制剂具有平均高 1.5 倍的比活性（活性按蛋白质量归一化），这归因于在生产过程中 CO 结构得到了更好的保护，因为蛋白浓度大大提高。我们得出结论，BDD-FVIII 的密码子优化导致其表达显著增加，并且不影响结构-功能特性。
