Abstract：
Based on observations indicating that the γ-carboxylase enzyme has a lower affinity for the protein C (PC) propeptide and that the γ-carboxylase region in the PC propeptide has a higher net charge, expression of recombinant chimeric factor IX (FIX) equipped with the PC propeptide was studied. The prepropeptide of FIX was replaced with that of PC by SOEing PCR and after cloning, recombinant pMT-prepro PC/FIX was transfected into insect Drosophila S2 cells. The expression and activity of expressed FIX were analyzed employing antigen and activity analyses 72 h of post-induction with copper. Higher secretion (1.2 fold) and activity (1.6 fold) levels were observed for chimeric prepro- PC/FIX in relation to wild-type FIX. Furthermore, after barium citrate precipitation, the evaluation of fully γ-carboxylated FIX indicated that more than 51% of the total FIX produced with the PC prepropeptide was fully γ-carboxylated, representing a substantial improvement (twofold) over a system employing the native FIX propeptide in which 25% of the protein is fully γ-carboxylated. The data illustrated that the expression of FIX using the PC propeptide led to much higher fully γ-carboxylated material, which is preferred to FIX constructs tolerating the sequence for the native FIX propeptide expressed in heterologous S2 systems.
摘要：
根据观察表明γ-羧化酶对蛋白C（PC）前肽具有较低的亲和力，并且PC前肽中的γ-羧化酶区域具有较高的净电荷，研究了装配有PC前肽的重组嵌合因子IX(FIX)的表达。通过SOEingPCR将FIX的前肽替换为PC，克隆后，将重组pMT-preproPC/FIX转染到昆虫果蝇S2细胞中。在用铜诱导后72小时,使用抗原和活性分析分析表达的FIX的表达和活性。相对于野生型FIX,对于嵌合prepro-PC/FIX观察到更高的分泌(1.2倍)和活性(1.6倍)水平。此外，柠檬酸钡沉淀后，完全γ-羧化FIX的评估表明，使用PC前肽产生的总FIX的51％以上是完全γ-羧化的，代表了相对于使用天然FIX前肽的系统的实质性改进(两倍),其中25%的蛋白质是完全γ-羧化的。数据表明，使用PC前肽的FIX表达导致更高的完全γ-羧化材料，其优选耐受在异源S2系统中表达的天然FIX前肽的序列的FIX构建体。
