Purification and characterization of a new recombinant factor VIII (N8)
Purification and characterization of a new recombinant factor VIII (N8)
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DOI：
10.1111/j.1365-2516.2009.02135.x
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发表时间：
2010-03-01
期刊：
影响因子：
3.9
通讯作者：
Steenstrup, T. D.
中科院分区：
文献类型：
--
作者：
Thim, L.;Vandahl, B.;Steenstrup, T. D.
一种新的重组因子 VIII (FVIII) N8 已在中国仓鼠卵巢 (CHO) 细胞中产生。该分子由一条 88 kDa 的重链（包括 21 个氨基酸残基的截短 B 结构域）和一条 79 kDa 的轻链组成。两条链通过非共价相互作用结合在一起。四步纯化包括捕获、使用单克隆重组抗体的亲和纯化、阴离子交换层析和凝胶过滤。 N8的比凝血活性为8800-9800 IU mg-1。序列和质谱分析揭示了轻链的两种变体，对应于血浆 FVIII 中也已知的两种替代 N 端序列。纯化产物中存在重链的两种变体，即带有和不带有 B 结构域接头的两种变体。该接头在 N8 的凝血酶激活后被去除，从而呈现类似于血浆 FVIIIa 的激活的 FVIII (FVIIIa) 分子。 FVIII 的所有六种已知酪氨酸硫酸化均在 N8 中得到证实。轻链的 A3 和 C1 结构域中存在两个 N-连接糖基化，重链的 A1 结构域中存在两个 N-连接糖基化。大多数 N 连接聚糖是唾液酸化的双触角结构。 B 结构域接头区域中存在 O-糖基化位点。约 65% 的产品中该位点被双唾液酸化 GalNAc-Gal 结构糖基化。总之，目前的数据表明，N8 是一种纯净且特征良好的 FVIII 产品，其生化特性与其他 FVIII 产品相同。
A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg-1. Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.