Castro José M, Horn Daniel A, Pu Xinzhu, Lewis Karen A
Department of Chemistry and Biochemistry, Texas State University, 601 University Dr., San Marcos, TX, 78666, United States.
Department of Chemistry and Biochemistry, Texas State University, 601 University Dr., San Marcos, TX, 78666, United States.
Protein Expr Purif. 2017 Jun;134:147-153. doi: 10.1016/j.pep.2017.04.004. Epub 2017 Apr 8.
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 (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His-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.
构成La相关蛋白（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。为了更全面地描述LARP6亚家族的生物学功能，我们从两种硬骨鱼，即剑尾鱼（Xiphophorus maculatus）和斑马鱼（Danio rerio）中重组表达了全长LARP6蛋白。使用N端His-SUMO标签的串联方法和迭代溶解度筛选试验来鉴定结构稳定的缓冲液成分，重组蛋白的产量提高到了>2 mg/L。通过有限蛋白酶解探测纯化的鱼类蛋白的结构域拓扑结构。鱼类蛋白含有一个内部的、抗蛋白酶的40 kDa结构域，该结构域比人类LARP6蛋白的可比结构域稳定得多。因此，鱼类蛋白是一个有利可图的模型系统，可用于研究该La相关蛋白家族的进化差异以及构成LARP6蛋白的结构域的结构和构象动力学。
