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首页> 外文期刊>Biochemistry >Trans protein splicing of cyanobacterial split inteins in endogenous and exogenous combinations
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Trans protein splicing of cyanobacterial split inteins in endogenous and exogenous combinations

机译：内源性和外源性组合中的蓝细菌分裂蛋白的反式蛋白剪接

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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.
机译：内含肽是自催化的蛋白质结构域，其在翻译后从蛋白质前体中切除，并在称为蛋白质剪接的过程中用肽键连接其侧翼区域。蓝细菌和纳米古菌的含内含肽的DNA聚合酶在其内含肽结构域中自然分裂成两个独立的基因。这种天然存在的分裂内含蛋白在反式中快速自缔合并重新组成蛋白分裂活性。在这里，我们分析了来自不同蓝藻的三种天然分裂的内含肽的体外蛋白剪接活性：颤藻，嗜热嗜热球菌和Nostoc sp。 PCC7120。将这些分裂的内含子的N和C末端半部分以9种组合进行混合，从而产生3种内源性（野生型）和6种外源性组合。尽管同源蛋白质之间有30-50％的序列差异，但在所有拆分内含肽组合中均检测到蛋白质剪接。剪接活性在多种条件下进行，包括变性剂和还原剂的存在以及高温，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。另外，发现铜离子在可逆的双锁反应中抑制反式剪接。我们对内源性和外源性组合中天然分裂的内含蛋白的比较分析证明了反式蛋白剪接元件的模块化及其强大的活性。这表明分裂内含子的一半与修饰内含子部分特异性的条件之间存在紧密的相互作用。这些结果促进了在体内或体外以及在中等或极端条件下，分裂内含子在蛋白质片段的受控组装中的生物技术应用。

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