A ribosomal misincorporation of Lys for Arg in human triosephosphate isomerase expressed in Escherichia coli gives rise to two protein populations.
A ribosomal misincorporation of Lys for Arg in human triosephosphate isomerase expressed in Escherichia coli gives rise to two protein populations.
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DOI：
10.1371/journal.pone.0021035
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Gómez Puyou A
中科院分区：
文献类型：
--
作者：
Aguirre B;Costas M;Cabrera N;Mendoza-Hernández G;Helseth DL Jr;Fernández P;de Gómez-Puyou MT;Pérez-Montfort R;Torres-Larios A;Gómez Puyou A
我们之前观察到，在大肠杆菌中表达的人类同型二聚体三磷酸酯异构酶（HsTIM）在纯化后表现出明显的同质性，表现出两种明显不同的热转变。对这一现象的详细研究表明，这些制剂含有两种蛋白质；一个具有预期的理论质量，而另一个的质量低28 Da。在3 M尿素中，用粒径排斥层析法分离这两个蛋白。串联质谱分析（LC/ESI-MS/MS）结果表明，这两种蛋白均与HsTIM相对应。在一定的生长条件下，这两种蛋白质的含量几乎相等。它们具有催化活性，但在分子质量、热稳定性、对尿素和蛋白酶k的敏感性方面存在差异。对人类TIM基因核苷酸的分析显示，存在6个在大肠杆菌中不常用的密码子。我们检查了它们是否与这两种蛋白质的形成有关。我们发现，在含有trna编码基因的菌株中，该酶的表达含有额外的拷贝，这些基因编码trna经常限制异种蛋白（Arg, Ile, Leu）的翻译，以及两个连续罕见的Arg密码子（位置98和99）的沉默突变，导致只产生更稳定的蛋白。LC/ESI-MS/MS进一步分析表明，28 Da的质量差是由于99位的赖氨酸取代了Arg残基。总的来说，我们的工作表明，在相同的细胞环境中共存的两种具有不同生化和生物物理特性的蛋白质是从相同的核苷酸序列框架翻译而来的。
We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.
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影响因子：
3.9
作者：
Freeland, SJ;Hurst, LD
通讯作者：
Hurst, LD
影响因子：
1.6
作者：
McNulty, DE;Claffee, BA;Kane, JF
通讯作者：
Kane, JF
影响因子：
1.6
作者：
Day, AJ;Aplin, RT;Willis, AC
通讯作者：
Willis, AC
影响因子：
7.7
作者：
Dittmar, KA;Sorensen, MA;Pan, T
通讯作者：
Pan, T
影响因子：
5.6
作者：
Calderone, TL;Stevens, RD;Oas, TG
通讯作者：
Oas, TG