Modulation of intein activity by its neighboring extein substrates
Modulation of intein activity by its neighboring extein substrates
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DOI：
10.1073/pnas.0904366106
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发表时间：
2009-07-07
影响因子：
11.1
通讯作者：
Belfort, Marlene
中科院分区：
文献类型：
--
作者：
Amitai, Gil;Callahan, Brian P.;Belfort, Marlene
内含肽是一类广泛存在于宿主蛋白中的自剪接蛋白催化剂。内含肽和分裂宿主蛋白（外蛋白）之间的进化和功能关系在很大程度上是未知的。为了探测关联，我们开发了基于FRET的体内和体外内含肽测定。FRET测定报告内含肽从其N-末端外显肽裂解。应用此检测随机外显肽库，我们表明，外显肽基板接壤的内含肽的性质可以深刻影响内含肽的活性。在N-和C-末端外显蛋白中接近内含肽-剪接接合点的残基可使N-末端切割速率加速> 4倍或使切割减弱1，000倍，两者均导致自我剪接效率受损。外显肽效应的存在和大小需要考虑最大限度地提高内含肽在生物技术应用中的效用，并且它们预测了自然界中内含肽整合位点的偏差。
Inteins comprise a large family of phylogenetically widespread self-splicing protein catalysts that colonize diverse host proteins. The evolutionary and functional relationship between the intein and the split-host protein, the exteins, is largely unknown. To probe an association, we developed an in vivo and in vitro intein assay based on FRET. The FRET assay reports cleavage of the intein from its N-terminal extein. Applying this assay to randomized extein libraries, we show that the nature of the extein substrate bordering the intein can profoundly influence intein activity. Residues proximal to the intein-splicing junction in both N- and C-terminal exteins can accelerate the N- terminal cleavage rate by > 4-fold or attenuate cleavage by 1,000-fold, both resulting in compromised self-splicing efficiency. The existence and the magnitude of extein effects require consideration for maximizing the utility of inteins in biotechnological applications, and they predict biases in intein integration sites in nature.