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通过RadA分裂内含肽在蛋白质中高效生成酰肼

Efficient Generation of Hydrazides in Proteins by RadA Split Intein.

作者信息

Liu Jun, Ekanayake Oshini, Santoleri Dominic, Walker Kelsi, Rozovsky Sharon

机构信息

Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA.

Department of Pharmaceutical Chemistry, University of California San Francisco, 555 Mission Bay Boulevard South, San Francisco, CA, 94158, USA.

出版信息

Chembiochem. 2020 Feb 3;21(3):346-352. doi: 10.1002/cbic.201900160. Epub 2019 Oct 11.

Abstract

Protein C-terminal hydrazides are useful for bioconjugation and construction of proteins from multiple fragments through native chemical ligation. To generate C-terminal hydrazides in proteins, an efficient intein-based preparation method has been developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method has been expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. It is expected that this versatile preparation method will expand the utilization of protein C-terminal hydrazides in protein preparation and modification.

摘要

蛋白质C末端酰肼可用于生物共轭以及通过天然化学连接从多个片段构建蛋白质。为了在蛋白质中生成C末端酰肼,已开发出一种基于内含肽的高效制备方法,该方法利用硫醇和肼来加速瞬态硫酯中间体的形成及随后的肼解反应。这种方法不仅提高了产率,还改善了生物相容性。通过使用嗜热栖热菌RadA分裂内含肽,该方法的适用范围得到了扩展,它能够在切割位点之前容纳广泛的外显肽残基。使用分裂RadA可最大限度减少体内内含肽N端的过早切割,并能控制内含肽N端切割反应的起始。预计这种通用的制备方法将扩大蛋白质C末端酰肼在蛋白质制备和修饰中的应用。

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