Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli
Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli
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DOI：
10.1016/s1046-5928(02)00610-1
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发表时间：
2003-02-01
影响因子：
1.6
通讯作者：
Kane, JF
中科院分区：
文献类型：
--
作者：
McNulty, DE;Claffee, BA;Kane, JF
在大肠杆菌中，CGG是一种罕见的精氨酸密码子，在所有大肠杆菌的mRNAs中出现的频率为0.54%，当精氨酸残基被编码时，出现频率为9.8%。当在高表达的重组mRNA中大量存在或成簇存在时，稀有密码子可能会导致表达问题，影响产物产量和翻译保真度。单纯疱疹病毒2型(HSV-2)N端多组氨酸标记的p27蛋白酶域的编码区含有11个这种稀有的精氨酸密码子，其中3个在蛋白的C末端附近串联出现。重组蛋白在大肠杆菌中表达时，经SDS-PAGE凝胶分析，大部分重组蛋白的表观分子量为31 kDa，比预测的大3 kDa。对蛋白质和多肽片段进行了详细的生化分析，并用Edman和MS/MS测序方法进行了鉴定。分离到两个主要物种，包括三联体簇中第二和第三个CGG密码子上的+1个移码事件。翻译在错义框架中进行到下一个终止密码子。此外，发现显著水平的谷氨酰胺错配成精氨酸，这表明第二碱基将CGG误读为CAG。共表达ARGX基因，编码CGG密码子的同源tRNA，在很大程度上消除了移码和误结合事件，并将正品的表达水平提高了7倍。我们的结论是，通过共表达ARGX基因来补充稀有的精氨酸基tRNACGG水平可以在很大程度上减轻大肠杆菌中存在的CGG密码子偏差，从而允许高效和准确地翻译异源基因产物。(C)2002年埃尔塞维尔科学公司(美国)。版权所有。
In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E coli mRNAs or 9.8% when an arginine residue is encoded for. When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity. The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein. When expressed in E coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted. Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches. Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster. Translation proceeded in the missense frame to the next termination codon. In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG. Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold. We conclude that supplementation of the rare arginyl tRNACGG levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E coli, allowing for efficient and accurate translation of heterologous gene products. (C) 2002 Elsevier Science (USA). All rights reserved.