Watanabe Miki, Miyazono Ken-ichi, Tanokura Masaru, Sawasaki Tatsuya, Endo Yaeta, Kobayashi Ichizo
Department of Medical Genome Sciences, Graduate School of Frontier Science, University of Tokyo, Tokyo, Japan.
Methods Mol Biol. 2010;607:149-60. doi: 10.1007/978-1-60327-331-2_13.
Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.
对于那些对细胞有毒且无法在体内大量制备的蛋白质来说，其结构测定一直颇具难度。这些蛋白质，即便在生物学上极具研究价值，在结构基因组学项目中往往也未得到充分表征。它们的无细胞合成能够规避毒性问题。在各种无细胞系统中，基于小麦胚芽的系统因其以下几点而格外引人关注：（1）由于基因置于植物翻译信号之下，其在细菌宿主中的毒性表达会降低。（2）它几乎没有密码子偏好，尤其是对甲硫氨酸和硒代甲硫氨酸（SeMet）的区分极小，这使得通过SAD和MAD方法轻松制备用于晶体结构测定的硒代甲硫氨酸化蛋白质成为可能。（3）翻译与转录解偶联，这样一来，如果翻译产物对DNA及其转录存在毒性，也能够被规避。我们已经证明，基于小麦胚芽的无细胞蛋白质合成对于一种4碱基切割限制酶的X射线晶体学研究很有用，这类限制酶预计对所有保留基因组的细胞形式都具有很强的毒性。我们关于其结构的报告代表了首次使用基于小麦胚芽的无细胞蛋白质表达系统过表达的蛋白质通过X射线晶体学进行结构测定的报告。当成本不成问题时，这将是细胞毒性蛋白质的首选方法。当晶体结构测定技术发展到仅需极少量蛋白质时，它的应用将会变得普及。
