Schoner B E, Belagaje R M, Schoner R G
Proc Natl Acad Sci U S A. 1986 Nov;83(22):8506-10. doi: 10.1073/pnas.83.22.8506.
A synthetic two-cistron expression system was constructed for the high-level expression of eukaryotic genes in Escherichia coli. This system was designed to overcome translational inhibition of mRNAs containing eukaryotic sequences. The first cistron in this system is a 31-base A + T-rich synthetic sequence that provides for efficient translation initiation. The second cistron contains the protein coding sequence for the eukaryotic gene. Insertion of the first cistron between the 5' untranslated region of the mRNA and the protein coding region separates the two and thereby potentially minimizes the formation of local secondary structures that might prevent ribosomes from binding and initiating translation. The 31-base cistron contains three nonsense codons (TAA), one in each of the three translational reading frames, and an 8-base Shine-Dalgarno sequence that is complementary to the 3' end of the 16S rRNA. The effects of translation of the first cistron in all three reading frames on the expression of the second cistron was examined. The most efficient expression of the second cistron seemed to occur when the stop codon that terminates translation of the first cistron is located 3' to the Shine-Dalgarno sequence and close to the AUG start codon for the second cistron. When the Shine-Dalgarno sequence was deleted from the first cistron, no detectable expression of the second cistron was observed. This two-cistron system has been used to express the gene encoding methionylalanyl bovine growth hormone with its native codons and the gene encoding methionyl human growth hormone at a level greater than 20% of total cell protein. In the case of human growth hormone, we show that the amount of gene product is not significantly diminished by placing a "functional" first cistron in front of a gene that can be expressed without a cistron.
构建了一种合成双顺反子表达系统，用于在大肠杆菌中高效表达真核基因。该系统旨在克服对含有真核序列的mRNA的翻译抑制。此系统中的第一个顺反子是一个富含31个碱基的A + T的合成序列，可提供有效的翻译起始。第二个顺反子包含真核基因的蛋白质编码序列。将第一个顺反子插入mRNA的5'非翻译区和蛋白质编码区之间，将两者分开，从而可能使可能阻止核糖体结合和起始翻译的局部二级结构的形成最小化。31个碱基的顺反子包含三个无义密码子（TAA），在三个翻译阅读框中各有一个，以及一个与16S rRNA的3'末端互补的8个碱基的Shine-Dalgarno序列。研究了第一个顺反子在所有三个阅读框中的翻译对第二个顺反子表达的影响。当终止第一个顺反子翻译的终止密码子位于Shine-Dalgarno序列的3'端且靠近第二个顺反子的AUG起始密码子时，第二个顺反子的表达似乎最有效。当从第一个顺反子中删除Shine-Dalgarno序列时，未观察到第二个顺反子的可检测表达。这个双顺反子系统已被用于以天然密码子表达编码甲硫氨酰丙氨酰牛生长激素的基因以及编码甲硫氨酰人生长激素的基因，其表达水平超过总细胞蛋白的20％。就人生长激素而言，我们表明，通过在无需顺反子即可表达的基因前放置一个“功能性”第一个顺反子，基因产物的量不会显著减少。
