Motohashi Ken, Okegawa Yuki
Department of Bioresources and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.
Department of Bioresources and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.
Protein Expr Purif. 2014 Sep;101:152-6. doi: 10.1016/j.pep.2014.07.001. Epub 2014 Jul 10.
Plant redox-related proteins were overexpressed using a genetic codon substitution downstream of the translation initiation codon. This method significantly improved recombinant protein expression levels of Arabidopsis chloroplastic thioredoxins and cytosolic nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (E.C. 1.8.1.9) in Escherichia coli. Using these proteins, the in vitro chloroplastic thioredoxins-reduction system was reconstituted in an NADPH-dependent manner. This system could convert the five classes of chloroplastic Arabidopsis thioredoxins and two chloroplastic Spinach thioredoxins to their reduced forms, independent of dithiothreitol and the photosynthetic electron transport system.
通过在翻译起始密码子下游进行遗传密码子替换，使植物氧化还原相关蛋白过表达。该方法显著提高了拟南芥叶绿体硫氧还蛋白和大肠杆菌中胞质烟酰胺腺嘌呤二核苷酸磷酸（NADPH）依赖性硫氧还蛋白还原酶（E.C. 1.8.1.9）的重组蛋白表达水平。利用这些蛋白，以NADPH依赖性方式重建了体外叶绿体硫氧还蛋白还原系统。该系统可以将五类拟南芥叶绿体硫氧还蛋白和两类菠菜叶绿体硫氧还蛋白转化为还原形式，而不依赖于二硫苏糖醇和光合电子传递系统。
