Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues.
Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues.
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DOI：
10.1074/jbc.m112.433094
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发表时间：
2013-03-01
期刊：
影响因子：
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通讯作者：
Perler F
中科院分区：
文献类型：
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作者：
Cheriyan M;Pedamallu CS;Tori K;Perler F
背景：内含肽是蛋白质成熟的机器，使技术的调控酶和蛋白质半合成。结果：用新的遗传选择的外显蛋白实现了稳健的剪接。结论：与普遍持有的看法相反，天然外显蛋白不是最快的剪接底物。意义：天然前体平衡外显肽和内含肽选择性压力。特异性研究为识别新的内含肽插入位点提供了一个预测性的标尺。内含肽是天然存在的间插序列，其催化蛋白质剪接反应，导致内含肽切除和侧翼多肽（外显肽）与天然肽键的连接。内含肽在一个高度保守的折叠内显示出多种催化机制，该折叠与刺猬蛋白共享。内含肽的不寻常的化学性质提供了强大的生物技术工具，用于控制剪接后的酶功能，并允许不同来源的肽以特定的、时间限定的方式偶联。紧邻内含肽侧翼的外显肽序列影响剪接，并且可以定义为内含肽底物。由于所有可能的侧翼序列的巨大潜在复杂性，研究内含肽底物特异性一直是困难的。因此，我们开发了一种剪接依赖性卡那霉素抗性的遗传选择，当紧邻内含肽插入位点侧翼的6个氨基酸被随机化时，没有显著的偏倚。我们应用这种选择来检查Nostoc punctiforme Npu DnaE内含肽侧翼残基的序列空间，并发现该内含肽有效地剪接比以前认为的更宽范围的序列，几乎没有N-外显肽特异性，只有两个重要的C-外显肽位置。新选择的外显肽序列足以促进三种不相关蛋白质的剪接，证实了特异性数据的可推广性，并为任何靶点定义了新的潜在插入位点。动力学分析表明，剪接速率与选定的外显蛋白一样快或更快的天然外显蛋白，驳斥了过去的假设，自然选择的侧翼外显蛋白序列是最佳的剪接。
Background: Inteins are protein maturation machines that enable technologies for regulating enzymes and protein semisynthesis. Results: Robust splicing was achieved with novel genetically selected exteins. Conclusion: In contrast to commonly held perceptions, the natural extein was not the fastest splicing substrate. Significance: Natural precursors balance extein- and intein-selective pressures. Specificity studies provide a predictive rubric for identifying new intein insertion sites. Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.