Department of Chemistry, Frick Laboratory, Princeton University, Princeton, NJ 08544.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461.
Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):8538-8543. doi: 10.1073/pnas.1701083114. Epub 2017 Jul 24.
The protein -splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an "extein dependence," whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting "promiscuous" inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins.
天然分裂内含肽的蛋白质剪接（PTS）活性在化学生物学和生物技术中得到了广泛应用。然而，目前使用的天然分裂内含肽存在“外显肽依赖性”，即剪接连接处周围的残基强烈影响剪接效率，限制了许多基于 PTS 的方法的通用性。为了解决这个问题，我们描述了一种基于机制的蛋白质工程方法，该方法赋予超快 DnaE 分裂内含肽最小的外显肽依赖性。结果表明，这些“多功能”内含肽是用于蛋白质环化和蛋白质半合成的优良试剂，后者通过修饰天然细胞染色质得到了说明。这里报道的多功能内含肽提高了现有 PTS 方法的适用性，并且应该能够为未来将多功能性工程到其他天然分裂内含肽的努力提供支持。
