An Atypical Naturally Split Intein Engineered for Highly Efficient Protein Labeling
An Atypical Naturally Split Intein Engineered for Highly Efficient Protein Labeling
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DOI：
10.1002/anie.201307969
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发表时间：
2014-01-27
影响因子：
16.6
通讯作者：
Mootz, Henning D.
中科院分区：
文献类型：
--
作者：
Thiel, Ilka V.;Volkmann, Gerrit;Mootz, Henning D.
由分裂内含肽催化的蛋白质转拼是一种从两个独立部分组装多肽主链的强大技术。然而，具有强大效率的分裂内含子和适合肽合成的短片段很少见，并且大多是人工创造的。从宏基因组数据中鉴定并表征了新型分裂内含肽 AceL-TerL。它代表了第一个自然发生的非典型分裂内含肽。仅包含 25 个氨基酸的 N 端片段是迄今为止最短的天然内含肽片段，并且易于使用荧光标记进行化学合成。在低温下观察到最佳的蛋白质转拼活性。通过定向蛋白质进化选择了进一步改进的突变体。工程化内含肽变体的速率提高了 50 倍，在化学标记多种蛋白质方面表现出前所未有的效率。这些内含肽应该被证明是蛋白质半合成和其他内含肽相关生物技术应用的有价值的工具。
Protein trans-splicing catalyzed by split inteins is a powerful technique for assembling a polypeptide backbone from two separate parts. However, split inteins with robust efficiencies and short fragments suitable for peptide synthesis are rare and have mostly been artificially created. The novel split intein AceL-TerL was identified from metagenomic data and characterized. It represents the first naturally occurring, atypically split intein. The N-terminal fragment of only 25 amino acids is the shortest natural intein fragment to date and was easily amenable to chemical synthesis with a fluorescent label. Optimal protein trans-splicing activity was observed at low temperatures. Further improved mutants were selected by directed protein evolution. The engineered intein variants with up to 50-fold increased rates showed unprecedented efficiency in chemically labeling of a diverse set of proteins. These inteins should prove valuable tools for protein semi-synthesis and other intein-related biotechnological applications.