Doering Christopher B, Healey John F, Parker Ernest T, Barrow Rachel T, Lollar Pete
Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, USA.
J Biol Chem. 2004 Feb 20;279(8):6546-52. doi: 10.1074/jbc.M312451200. Epub 2003 Dec 1.
Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.
血液凝固因子VIII具有名为A1 - A2 - B - ap - A3 - C1 - C2的结构域结构。人因子VIII在正常血浆中的浓度较低，同样，使用转基因表达技术在体外和体内的产量也很低。在哺乳动物细胞培养中，缺失B结构域的猪因子VIII的异源表达显著高于缺失B结构域的人或鼠因子VIII。构建了新型人/猪杂交因子VIII分子，以鉴定赋予高水平表达的猪因子VIII结构域。含有猪因子VIII A1和ap - A3结构域的人/猪杂交因子VIII构建体的表达水平与重组猪因子VIII相当。仅含有猪A1结构域的杂交构建体的表达水平介于人和猪因子VIII之间，而含有猪ap - A3结构域的杂交构建体的表达水平与人因子VIII相当。此外，含有猪因子VIII A1和ap - A3结构域序列的鼠/猪杂交因子VIII构建体的表达水平显著高于重组鼠因子VIII。因此，猪A1和ap - A3结构域对于与猪因子VIII相关的高水平表达是必要且充分的。代谢放射性标记实验表明，高水平表达归因于分泌效率的提高。
