Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2.
FEBS Lett. 2012 Jan 2;586(1):79-84. doi: 10.1016/j.febslet.2011.11.028. Epub 2011 Dec 3.
Site-specific protein cleavage is a ubiquitous process in cellular protein metabolism, yet molecular tools to provide control of protein cleavage inside living cells remain scarce. Here, we show that the C-terminal intein fragment of the non-canonical Ssp (Synechocystis sp. PCC6803) DnaB S1 split-intein can be used as a site-specific protease for in vivo protein cleavage both in bacterial and eukaryotic cells. Mutagenesis data indicate a broad tolerance of the intein-derived protease (IP) toward the amino acid upstream of the cleavage site. Furthermore, deletion studies reveal that the recognition sequence for the IP can be as short as ten amino acids. The structural features underlying the cleavage reaction preclude unintended proteolysis of endogenous proteins, thus ensuring that negative effects on cell viability are minimal.
位点特异性蛋白切割是细胞内蛋白质代谢中普遍存在的过程，但提供在活细胞内控制蛋白切割的分子工具仍然稀缺。在这里，我们表明，非典型 Ssp（集胞藻 6803）DnaB S1 分裂内含子的 C 末端内含子片段可用作细菌和真核细胞中体内蛋白切割的位点特异性蛋白酶。突变数据表明，内含子衍生蛋白酶（IP）对切割位点上游的氨基酸具有广泛的耐受性。此外，缺失研究表明，IP 的识别序列可以短至十个氨基酸。切割反应的结构特征排除了对内源性蛋白质的非预期蛋白水解，从而确保对细胞活力的负面影响最小。
