Ohashi Yoshiaki, Yamashiro Akiko, Washio Takanori, Ishii Nobuyoshi, Ohshima Hideyuki, Michishita Tetsuko, Tomita Masaru, Itaya Mitsuhiro
Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
Gene. 2005 Feb 28;347(1):11-9. doi: 10.1016/j.gene.2004.11.027. Epub 2005 Jan 4.
The translation start site, immediately downstream from the start codon, is a dominant factor for gene expression in Escherichia coli. At present, no method exists to improve the expression level of cloned genes, since it remains difficult to find the best codon combination within the region. We determined the expression parameters that correspond to all sense codons within the first four codons using GFPuv which encodes a derivative of green fluorescent protein. Using a genetic algorithm (GA)-based computer program, these parameters were incorporated in a simple, static model for the prediction of translation efficiency, and optimized to the expression level for 137 randomly isolated GFPuv genes. The calculated initial translation index (ITI), also proven for the DsRed2 gene that encodes a red fluorescent protein, should provide a solution to overcome the gene expression problem in cloned genes whose expression is often inherently blocked at the translation process. The proposed method facilitates heterologous protein production in E. coli, the most commonly used host in biological and industrial fields.
翻译起始位点位于起始密码子下游紧邻处，是大肠杆菌中基因表达的一个主导因素。目前，尚无提高克隆基因表达水平的方法，因为在该区域内仍然难以找到最佳密码子组合。我们使用编码绿色荧光蛋白衍生物的GFPuv确定了前四个密码子内所有有义密码子对应的表达参数。利用基于遗传算法（GA）的计算机程序，将这些参数纳入一个简单的静态模型中以预测翻译效率，并针对137个随机分离的GFPuv基因的表达水平进行优化。计算得到的初始翻译指数（ITI），在编码红色荧光蛋白的DsRed2基因中也得到了验证，应该能为克服克隆基因在翻译过程中常常固有受阻的基因表达问题提供一个解决方案。所提出的方法有助于在大肠杆菌（生物和工业领域最常用宿主）中进行异源蛋白生产。
