High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli
High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli
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DOI：
10.1006/jmbi.1996.0524
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发表时间：
1996-10-04
影响因子：
5.6
通讯作者：
Oas, TG
中科院分区：
文献类型：
--
作者：
Calderone, TL;Stevens, RD;Oas, TG
真核基因在大肠杆菌中的表达是现代科学中最常用的工具之一。精氨酸密码子阿加是真核生物基因中常见的密码子，但在大肠杆菌中特别罕见。杆菌我们在这里报告36至42%的赖氨酸在三个阿加密码子的错误掺入一个良好表达的蛋白质。这种错误掺入产生了一种蛋白质，其电喷雾质谱（ESMS）在预期质量（M）、M-28、M-56和M-84处显示峰，强度分别为总强度的34.5（+/-0.7）、37.5（+/-1.1）、21.2（+/-1.7）和6.6（+/-0.5）%。将所有三个阿加密码子或最靠近基因3'端的两个密码子替换为更常见的CGC精氨酸密码子，得到具有单一ESMS峰的蛋白质。这些研究表明，在重组蛋白的稀有密码子处氨基酸的错误掺入可能远远高于先前所认为的。(C)1996年学术出版社
The expression of eukaryotic genes in Escherichia coli is one of the most frequently used tools of modern science. The arginine codon AGA is a common codon in eukaryotic genes but is particularly rare in E. coli. We report here 36 to 42% misincorporation of lysine at three AGA codons in a well-expressed protein. This misincorporation yields a protein whose electrospray mass spectrum (ESMS) shows peaks at the expected mass (M), M-28, M-56 and M-84 with intensities representing 34.5(+/-0.7), 37.5(+/-1.1), 21.2(+/-1.7) and 6.6(+/-0.5) % of the total intensity, respectively. Replacement of either all three AGA codons or the two closest to the 3' end of the gene by the more common CGC arginine codon gave a protein with a single ESMS peak.Misincorporation could also be eliminated by the co-expression of the tRNA(UCU)(Arg) gene, argU. These studies demonstrate that misincorporation of amino acids at rare codons of recombinant proteins can be far higher than previously thought. (C) 1996 Academic Press Limited