Ligation of Expressed Protein α-Hydrazides via Genetic Incorporation of an α-Hydroxy Acid
Ligation of Expressed Protein α-Hydrazides via Genetic Incorporation of an α-Hydroxy Acid
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DOI：
10.1021/cb300020s
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发表时间：
2012-06-01
影响因子：
4
通讯作者：
Liu, Lei
中科院分区：
文献类型：
--
作者：
Li, Yi-Ming;Yang, Mai-Yun;Liu, Lei
表达的蛋白质连接在合成肽和重组蛋白质之间架起了差距，从而显著增加了化学合成蛋白质的大小和复杂性。虽然基于内含肽的表达蛋白连接方法已被广泛应用于这方面，但新的表达蛋白连接方法的开发可以提高蛋白半合成的灵活性和能力。在这项研究中，一个新的替代版本的表达蛋白连接的开发相结合，最近开发的技术，酰肼为基础的肽连接和遗传密码扩展。与以前的基于内含肽的表达蛋白连接方法相比，新方法不需要使用蛋白剪接技术，并且产生重组蛋白α-酰肼作为连接中间体，其比蛋白α-硫酯更化学稳定。此外，利用M. Barkeri显示出表达重组蛋白骨架氧代酯的改进性能。以HdeA蛋白为模型蛋白，我们证明了基于酰肼的方法可以用于合成具有正确折叠结构和完整生物活性的蛋白质。因为PylRS-tRNA（CUA）（pyl）系统与原核和真核细胞都相容，所以本文提出的策略可以容易地扩展以操纵哺乳动物细胞中产生的蛋白质。新的基于酰肼的方法还可以通过允许更灵活地选择连接位点来补充基于内含肽的表达蛋白连接方法。
Expressed protein ligation bridges the gap between synthetic peptides and recombinant proteins and thereby significantly increases the size and complexity of chemically synthesized proteins. Although the intein-based expressed protein ligation method has been extensively used in this regard, the development of new expressed protein ligation methods may improve the flexibility and power of protein semisynthesis. In this study a new alternative version of expressed protein ligation is developed by combining the recently developed technologies of hydrazide-based peptide ligation and genetic code expansion. Compared to the previous intein-based expressed protein ligation method, the new method does not require the use of protein splicing technology and generates recombinant protein alpha-hydrazides as ligation intermediates that are more chemically stable than protein alpha-thioesters. Furthermore, the use of an evolved mutant pyrrolysyl-tRNA synthetase (PylRS), ACPK-RS, from M. barkeri shows an improved performance for the expression of recombinant protein backbone oxoesters. By using HdeA as a model protein we demonstrate that the hydrazide-based method can be used to synthesize proteins with correctly folded structures and full biological activity. Because the PylRS-tRNA(CUA)(pyl) system is compatible with both prokaryotic and eukaryotic cells, the strategy presented here may be readily expanded to manipulate proteins produced in mammalian cells. The new hydrazide-based method may also supplement the intein-based expressed protein ligation method by allowing for a more flexible selection of ligation site.