经密码子优化的葡萄糖醛酸酶在E.coli中的表达

The Effect of mRNA Secondary Structure on α-glucuronidase Expression

对α-葡萄糖醛酸酶基因(AguA)的稀有密码子进行了优化,经过稀有密码子优化的基因在pTrc99A载体中由于二级结构稳定性的增加使得目的基因的表达水平比原始基因还要低.在权衡几个载体后使用本实验室构建的热激载体pAT,可以降低mRNA二级结构自由能(△g),得到了α-葡萄糖醛酸酶的高表达,结果充分说明了mRNA二级结构的自由能对外源基因的表达的影响.

It is well known that the amount of heterogeneous proteins produced by E. coli cells depends on various factors, such as the copy number and stability of the expression plasmid in the cells, the efficiency of translation initiation ,and the stability of the synthesized protein in E. coli cells. The efficiency of translation initiation in E. coli is determined by the stabilization of mRNA 5' end secondary structure and the frequency of rare condons' usage. In this study we optimized rare condons of α-glucuronidase, but the data showed that the expression of targeted gene is lower than that of original gene. The optimization of rare condons cause it more steady than the original gene in plasmid pTrc99A. After compared the free energy of several plasmids on hand we selected the heat shock plasmid pAT which was constrcted in the lab. The free energy of the genes cloned in this plasmid is decreased greatly and the expression level of target protein is increased largly. It is concluded that the free energy of mRNA 5' end secondary structure plays a great role in the expression level of foreign gene in E. coli.