Heterologous expression of the lipid transfer protein CERT increases therapeutic protein productivity of mammalian cells
Heterologous expression of the lipid transfer protein CERT increases therapeutic protein productivity of mammalian cells
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DOI：
10.1016/j.jbiotec.2009.02.014
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发表时间：
2009-04-20
影响因子：
4.1
通讯作者：
Kaufmann, Hitto
中科院分区：
文献类型：
--
作者：
Florin, Lore;Pegel, Antje;Kaufmann, Hitto
最近的研究表明，引入调节蛋白质转运或影响翻译后修饰的转基因可以进一步改善在哺乳动物细胞中生产治疗性蛋白质的工业过程。我们通过异源表达神经酰胺转移蛋白（CERT）来提高CHO细胞中治疗性蛋白质产量的研究是由最近发现CERT参与蛋白激酶D（PKD）依赖的蛋白质从高尔基体转运到质膜而开始的。我们通过稳定整合表达CERT野生型或CERT S132A的构建体来产生一组CHO DG44细胞系，赋予增加的脂质转移活性的突变体或模拟质粒。当在接种物悬浮培养物中生长时，表达异源CERT的CHO细胞表现出显著更高的治疗性蛋白HSA的比生产率。这种效应转化为在补料分批形式中显著增加的总体HSA滴度，其中细胞在化学成分确定的无血清培养基中生长。此外，我们可以表明，CERT也增强了单克隆抗体分泌在两个IgG生产细胞系具有不同的基础productivity.The数据证明了CERT工程的潜力，以改善哺乳动物细胞培养生产过程中，产生大量的治疗性蛋白质产品所需的质量。据我们所知，这是第一个研究表明，在哺乳动物细胞的高尔基复合体的重组蛋白分泌的瓶颈。(C)2009爱思唯尔有限公司版权所有。
Recent studies have demonstrated that the introduction of transgenes regulating protein transport or affecting post-translational modifications can further improve industrial processes for the production of therapeutic proteins in mammalian cells. Our study on improving therapeutic protein production in CHO cells by heterologous expression of the ceramide transfer protein (CERT) was initiated by the recent discovery that CERT is involved in protein kinase D (PKD)-dependent protein transport from the Golgi to the plasma membrane.We generated a set of CHO DG44 cell lines by stable integration of constructs expressing either CERT wild-type or CERT S132A, a mutant conferring increased lipid transfer activity, or a mock plasmid. CHO cells expressing heterologous CERT demonstrated significantly higher specific productivities of the therapeutic protein HSA when grown in inoculum suspension cultures. This effect translated into significantly increased overall HSA titers in a fed-batch format where cells are grown in chemically defined serum-free media. Furthermore, we could show that CERT also enhanced monoclonal antibody secretion in two IgG production cell lines with different basal productivities.The data demonstrate the potential of CERT engineering to improve mammalian cell culture production processes to yield high amounts of a therapeutic protein product of desired quality. To our knowledge, this is the first study showing a bottle neck in recombinant protein secretion at the Golgi complex in mammalian cells. (C) 2009 Elsevier B.V. All rights reserved.