人促红细胞生成素在大肠杆菌中融合高表达：稀有密码子对外源蛋白翻译速率的影响

本实验分别用两种融合表达载体（ｐＧＥＸ－２Ｔ及ｐＤＳ－６Ｈ）在大肠杆菌ＸＬ１－Ｂｌｕｅ中表达人促红细胞生成素（ｈ－ＥＰＯ）。结果发现，虽然ｈ－ＥＰＯ基因中大肠杆菌使用频率为０％或１％的密码子占密码子总量的１４．３％，但实验中却取得了较高的蛋白表达量。ｐＧＥＸ－２Ｔ和ｐＤＳ－６Ｈ中表达的融合蛋白（分子量：４４４００和２３８００）分别占细菌总蛋白的２９．７％和１７．５％。从而提示，一个基因中的密码子使用频率与翻译延长速率之间可能不是一种简单的对应关系。表达载体ｐＧＥＸ－２Ｔ中５＇融合基因长度达１３００ｂｐ，而ｐＤＳ－６Ｈ中仅为３６ｂｐ，而ｐＤＳ－６Ｈ但两者表达水平却无显著差异，因此作为翻译起始限制因素的翻译起始区的ＡＴＧ下游顺序仅需长约３６个核苷酸。

High fusion expression of human EPO in Escherichia coli: effect of rare codons on translation rate of exogenous protein

The human erythropoietin(h-EPO) cDNA was expressed in Escherichia coli(E.coli) by using two prokaryotic fusion expression vectors (pGEX-2T and pDS-6H). The results showed that the rare codons in h-EPO cDNA,whose frequency was 0 or 1 percent in E. coli, made up 14.3% of the total codon sequence, nevertheless, the relatively high expression level was obtained in the experiment. The expressed fusion proteins(molecular weight: 44 400 and 23 800) comprised 29.7% and 7.5% of the total cell protein. This suggests that there is no simple correspondence between the codon frequency and the translation rate of gene in E. coli. In pGEX-2T, the length of 5'-fusion gene is 1300 bp,yet in pDS-6H, it is only 36 bp while there is no marked expression difference between them. So, the sequence down stream from ATG of TI R(Translation initiation region) which confines translation to start only requires about 36 bp.