Trans protein splicing of cyanobacterial split inteins in endogenous and exogenous combinations
Trans protein splicing of cyanobacterial split inteins in endogenous and exogenous combinations
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DOI：
10.1021/bi0611762
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发表时间：
2007-01-09
期刊：
影响因子：
2.9
通讯作者：
Pietrokovski, Shmuel
中科院分区：
文献类型：
--
作者：
Dassa, Bareket;Amitai, Gil;Pietrokovski, Shmuel
内含肽是自催化蛋白质结构域，其在称为蛋白质剪接的过程中从蛋白质前体中切除并将其侧翼区域与肽键连接。蓝细菌和纳米古细菌的含内含肽的DNA聚合酶在其内含肽结构域天然地分裂成两个独立的基因。这种自然发生的分裂内含肽迅速自我关联和重组蛋白质剪接活动的反式。在这里，我们分析了在体外蛋白质剪接活动的三个自然分裂内含肽从不同的蓝藻：颤藻limnetica，Thermosynechococcus vulcanus，念珠藻属。PCC 7120。这些分裂内含肽的N-和C-末端的一半混合在9个组合中，导致3个内源性（野生型）和6个外源性组合。在所有的分裂内含肽组合中检测到蛋白质剪接，尽管同源蛋白质之间存在30-50%的序列变异。剪接活动进行了各种条件下，包括存在的变性剂和还原剂和高温，离子强度和粘度。尽管如此，在高浓度的盐（2 M）或尿素（6 M）中，特定组合的拼接效果明显优于其他组合。此外，发现铜离子在可逆双锁反应中抑制反式剪接。我们对内源性和外源性组合的天然分裂内含肽的比较分析表明了反式蛋白剪接元件的模块性及其强大的活性。这表明分裂内含肽半体之间的紧密相互作用和改变内含肽部分特异性的条件。这些结果促进了生物技术使用的分裂内含肽的蛋白质片段的控制组装在体内或体外，在温和或极端的条件下。
Inteins are autocatalytic protein domains that post-translationally excise from protein precursors and ligate their flanking regions with a peptide bond, in a process called protein splicing. Intein-containing DNA polymerases of cyanobacteria and nanoarchaea are naturally split into two separate genes at their intein domain. Such naturally occurring split inteins rapidly self-associate and reconstitute protein-splicing activity in trans. Here, we analyze the in vitro protein-splicing activity of three naturally split inteins from diverse cyanobacteria: Oscillatoria limnetica, Thermosynechococcus vulcanus, and Nostoc sp. PCC7120. N- and C-terminal halves of these split inteins were mixed in nine combinations, resulting in three endogenous (wild-type) and six exogenous combinations. Protein splicing was detected in all split-intein combinations, despite a 30-50% sequence variation between the homologous proteins. Splicing activity proceeded under a variety of conditions, including the presence of denaturants and reductants and high temperature, ionic strength, and viscosity. Still, in a high concentration of salt (2 M) or urea (6 M), specific combinations spliced significantly better than others. Additionally, copper ions were found to inhibit trans splicing in a reversible double-lock reaction. Our comparative analysis of naturally split inteins in endogenous and exogenous combinations demonstrates the modularity of trans protein-splicing elements and their robust activity. It suggests tight interactions between split-intein halves and conditions for modifying the specificity of intein parts. These results promote the biotechnological use of split inteins for controlled assembly of protein fragments either in vivo or in vitro and under moderate or extreme conditions.