Sonkoly Boglárka, Bardóczy Viola, Mészáros Tamás
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.
Methods Mol Biol. 2011;779:55-63. doi: 10.1007/978-1-61779-264-9_3.
In vitro functional studies of eukaryotic kinases are often constrained by the availability of pure and -enzymatically active kinase of interest. Though numerous proteins have been synthesized by cell-based systems, in vivo production of properly folded, eukaryotic proteins remains a challenging task. Current wheat-germ-based cell-free in vitro translation systems present a plausible alternative for protein synthesis since majority of eukaryotic proteins could be obtained in their native folded form with general protocols. The use of special in vitro translation vectors with ligation-independent cloning sites and cleavable affinity tags eliminates further bottlenecks of the protein producing procedure and makes this system a reasonable method for simultaneous generation of active kinases.
真核激酶的体外功能研究常常受到目标纯且具有酶活性的激酶可用性的限制。尽管基于细胞的系统已合成了众多蛋白质，但真核蛋白质在体内正确折叠的生产仍然是一项具有挑战性的任务。当前基于麦胚的无细胞体外翻译系统为蛋白质合成提供了一种可行的替代方案，因为通过通用方案大多数真核蛋白质能够以其天然折叠形式获得。使用具有不依赖连接的克隆位点和可切割亲和标签的特殊体外翻译载体消除了蛋白质生产过程中的进一步瓶颈，并使该系统成为同时生成活性激酶的合理方法。
