Nemoto Keiichirou, Nozawa Akira, Yamanaka Satoshi, Nomura Shunsuke, Kido Kohki, Sawasaki Tatsuya
Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan.
Methods Mol Biol. 2017;1621:113-120. doi: 10.1007/978-1-4939-7063-6_11.
The wheat germ cell-free protein synthesis system has a significant advantage for high-throughput production of eukaryotic multidomain proteins in a folded state. In this chapter, we describe two kinds of methods for performing autophosphorylation assay of plant receptor kinases (PRKs) by using the wheat cell-free system. One is an in vitro kinase assay performed using biotin-streptavidin affinity purification technology, and the other is a luminescence-based high-throughput assay for autophosphorylation analysis. We anticipate that our cell-free-based methods might facilitate the characterization of plant PRKs.
小麦胚无细胞蛋白质合成系统在高通量生产折叠状态的真核多结构域蛋白质方面具有显著优势。在本章中，我们描述了两种利用小麦无细胞系统进行植物受体激酶（PRK）自磷酸化测定的方法。一种是使用生物素-链霉亲和素亲和纯化技术进行的体外激酶测定，另一种是基于发光的自磷酸化高通量分析方法。我们预计，我们基于无细胞的方法可能有助于植物PRK的特性鉴定。
