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首页> 外文期刊>Biotechnology Progress >Role of Silent Gene Mutations in the Expression of Caprine Growth Hormone in Escherchia coli
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Role of Silent Gene Mutations in the Expression of Caprine Growth Hormone in Escherchia coli

机译：沉默基因突变在大肠杆菌中生长激素表达中的作用

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This report describes the strategy for overexpression of caprine growth hormone(cGH)gene of beetal goat in E.coli through introducing silent mutations in the 5'-end of the coding sequence.The silent mutations introduced were aimed at minimizing translation-inhibiting secondary structures in the mRNA.Free energies of the resultant mRNAs were calculated from the ribosomal binding site of mRNA to +24 base using the Mfold web server.The construct with native sequence did not show any expression,whereas introduction of the silent mutations had strong influence on the expression levels.Some constructs(pETcGH2-7)showed 12-30% expression of total cell proteins while some others(pETcGH8-16)showed 30 to 53% of total cell protein.Any variation in the amount of mRNA transcript for the various constructs,as determined by quantitative PCR,was not enough to suggest that the variable level of the gene expression was due to variation in the transcription levels.It appears that the expression levels are not always correlated with free-energy values of the secondary structures in the 5'-end region of the mRNA;instead some key silent nucleotide alterations at certain sites of 5'-end of the sequence reorganize the secondary structure in such a way that it has positive impact on translation without considerably altering the free-energy values.An empirical approach for determining the optimum 5'-end substitutions for hyperexpression of a recombinant protein thus seems necessary.
机译：本报告介绍了通过在编码序列的5'末端引入沉默突变来在大肠杆菌中过量表达山羊山羊的山羊生长激素（cGH）基因的策略。引入的沉默突变旨在最小化翻译抑制二级结构使用Mfold网络服务器从mRNA的核糖体结合位点到+24碱基计算得到的mRNA的自由能。具有天然序列的构建体未显示任何表达，而沉默突变的引入对MRNA的影响很大。某些构建体（pETcGH2-7）显示了总细胞蛋白的12-30％表达，而另一些构建体（pETcGH8-16）则显示了细胞总蛋白的30％至53％。通过定量PCR确定的构建体不足以表明基因表达的可变水平是由于转录水平的变化所致。并不总是与mRNA 5'端区域中二级结构的自由能值相关;相反，序列5'端某些位点的一些关键沉默核苷酸改变会重新组织二级结构，在不显着改变自由能值的情况下对翻译具有积极影响。因此，确定用于重组蛋白超表达的最佳5'-末端取代的经验方法似乎是必要的。

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