Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms
Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms
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DOI：
10.1016/j.pep.2017.04.004
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发表时间：
2017-06-01
影响因子：
1.6
通讯作者：
Lewis, Karen A.
中科院分区：
文献类型：
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作者：
Castro, Jose M.;Horn, Daniel A.;Lewis, Karen A.
组成La-Related Protein(LARP)超家族的RNA结合蛋白参与了从tRNA成熟到蛋白质合成调控等广泛的细胞功能。为了进一步研究LARP6亚家族的生物学功能，我们重组表达了两种硬骨鱼LARP6的全长蛋白，即斑点剑尾鱼和斑马鱼。利用N-末端His(6)-SUMO标签的串联方法和迭代溶解度筛选实验鉴定结构稳定的缓冲成分，重组蛋白的产量提高到>；2 mg/L。用有限的蛋白水解法对纯化的鱼蛋白的结构进行了研究。鱼蛋白含有一个内部的、抗蛋白酶的40 kDa结构域，比人类LARP6蛋白的可比结构域要稳定得多。因此，鱼类蛋白是一个有利可图的模型系统，可以在其中研究这一La相关蛋白家族的进化差异以及组成LARP6蛋白的结构域的结构和构象动力学。(C)2017 Elsevier Inc.保留所有权利。
The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Dario rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His(6)-SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein. (C) 2017 Elsevier Inc. All rights reserved.