A simplified reconstitution of mRNA-directed peptide synthesis: Activity of the epsilon enhancer and an unnatural amino acid
A simplified reconstitution of mRNA-directed peptide synthesis: Activity of the epsilon enhancer and an unnatural amino acid
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DOI：
10.1006/abio.2001.5329
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发表时间：
2001-10-01
影响因子：
2.9
通讯作者：
Blacklow, SC
中科院分区：
文献类型：
--
作者：
Forster, AC;Weissbach, H;Blacklow, SC
用易于提纯的成分进行重构，将极大地促进对翻译早期事件的研究。在这里，利用5个纯化的重组组氨酸标签的大肠杆菌起始和延伸因子，重组了大肠杆菌寡肽的合成。需要高纯度的核糖体才能产生对翻译因子有很强依赖性的产品。基于从编码四肽的信使核糖核酸中分离放射性标记的翻译产物，大约80%的多肽产物是全长的，剩下的20%是由于暂停或提前终止而产生的二肽和三肽产物。当mRNA中包含常用的epsilon(蛋白质合成起始增强子)序列时，寡肽的合成被增强。该系统结合了一种可选择的、大的、非天然的氨基酸，最终可能形成一种纯翻译展示技术的基础，用于模拟多肽配体和候选药物的定向进化。重组克隆可用于制备结构研究的起始因子和起始复合体，研究核糖体肽合成中的起始和延伸，以及筛选真细菌特异性药物。(C)2001年学术出版社。
The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs. (C) 2001 Academic Press.