期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 457, 期 1, 页码 77-82出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2014.12.082
关键词
Bioluminescence; Coelenterazine; Copepod luciferase; Mammalian expression; Real-time imaging
资金
- Bayer AG (Germany)
- Russian Science Foundation [14-14-01119]
- Russian Science Foundation [14-14-01119] Funding Source: Russian Science Foundation
Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 S-S bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of similar to 3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. (C) 2014 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据