BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies
BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies
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DOI：
10.1016/j.pep.2008.08.004
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发表时间：
2008-12-01
影响因子：
1.6
通讯作者：
Garcia, K. Christopher
中科院分区：
文献类型：
--
作者：
Dukkipati, Abhirarn;Park, Hyun Ho;Garcia, K. Christopher
杆状病毒介导的哺乳动物细胞基因转导（BacMam）是一种新兴的在哺乳动物细胞中快速表达重组蛋白的技术。我们构建了两个杆状病毒转移载体，将几个哺乳动物转录调控元件在哺乳动物细胞中的高水平蛋白表达所必需的。使用这些载体，我们表明，BacMam系统与293 GnTI（-）细胞系的组合，可用于生产毫克量的可溶性糖蛋白。此外，对于结晶试验，纯化的糖蛋白对EndoH处理敏感，导致大量连接的N-连接的糖基化的损失。此外，我们还表明，BacMam系统和293 GnTI（-）细胞系的组合可用于生产毫克量的GPCR-蛋白质配体复合物，适用于结晶试验。(c)2008年爱思唯尔公司All rights reserved.
Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. We constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high-level protein expression in mammalian cells. Using these vectors, we show that the BacMam system in combination with the 293 GnTI(-) cell line can be used for production of milligram quantities of soluble glycoproteins. Moreover, for crystallization trials, the purified glycoproteins are sensitive to EndoH treatment resulting in a loss of the bulk of the attached N-linked glycosylation. In addition, we also show that a combination of the BacMam system and 293 GnTI(-) cell line can be used for producing milligram quantities of a GPCR-protein ligand complex suitable for crystallization trials. (c) 2008 Elsevier Inc. All rights reserved.