Wang G, Liu N, Yang K
Institute of Microbiology, Chinese Academy of Sciences, Beijing.
Protein Expr Purif. 1995 Jun;6(3):284-90. doi: 10.1006/prep.1995.1037.
Regulation of the expression of prochymosin cDNA in Escherichia coli at the translational level was studied by mutating the regions between the Shine-Dalgarno (SD) sequence and the initiation codon and upstream of the SD signal. Results revealed that expression plasmids with a distance of 7-10 bp from SD to ATG have the potential to be expressed at higher levels. However, an approximately 20-fold variation in expression was observed with plasmids harboring different base composition but identical distance in the spacer. Analysis of the predicted secondary structure of ribosome binding sites (RBS) indicates that the control of expression by base composition is mediated by the secondary structure of the RBS. An unfolded state of the RBS is required for high expression. Therefore, a vector for enhanced translation can be designed and constructed via prediction of the secondary structure of the proposed RBS and mutagenesis. Based on this strategy, high-level expression of prochymosin, up to 39% of the total cellular proteins, was achieved. The 9-base sequence proposed by Olins and Rangwala as a translational enhancer did not exhibit an additive effect on prochymosin expression. This is probably because the affinity of the SD sequence used in this study to 16S rRNA is strong enough that no additional element is required to facilitate the formation of the translation initiation complex.
通过对夏因-达尔加诺（SD）序列与起始密码子之间以及SD信号上游区域进行突变，研究了大肠杆菌中凝乳酶原cDNA在翻译水平上的表达调控。结果表明，从SD到ATG距离为7-10个碱基对的表达质粒具有较高水平表达的潜力。然而，在间隔区距离相同但碱基组成不同的质粒中，观察到表达有大约20倍的差异。对核糖体结合位点（RBS）预测二级结构的分析表明，碱基组成对表达的控制是由RBS的二级结构介导的。RBS的未折叠状态是高表达所必需的。因此，可以通过预测拟议RBS的二级结构并进行诱变来设计和构建增强翻译的载体。基于此策略，实现了凝乳酶原的高水平表达，高达细胞总蛋白的39%。Olins和Rangwala提出的作为翻译增强子的9碱基序列对凝乳酶原表达没有表现出累加效应。这可能是因为本研究中使用的SD序列与16S rRNA的亲和力足够强，以至于不需要额外的元件来促进翻译起始复合物的形成。
