Engineering artificially split inteins for applications in protein chemistry:: Biochemical characterization of the split Ssp DnaB intein and comparison to the split Sce VMA intein
Engineering artificially split inteins for applications in protein chemistry:: Biochemical characterization of the split Ssp DnaB intein and comparison to the split Sce VMA intein
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DOI：
10.1021/bi051697
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发表时间：
2006-02-14
期刊：
影响因子：
2.9
通讯作者：
Mootz, HD
中科院分区：
文献类型：
--
作者：
Brenzel, S;Kurpiers, T;Mootz, HD
在蛋白质反式剪接中，内含子结构域分裂成两条多肽链，介导侧翼氨基酸序列、N-末端和C-末端外显子与天然肽键的连接。可以利用这一过程从两个单独制备的片段中组装蛋白质，例如用于核磁共振研究的同位素分段标记或结合化学和生物物理探针。分裂的内含子可以通过遗传手段人工产生；然而，纯化的内含素(N)和内含素(C)片段通常需要变性和复性处理才能折叠成活性内含子，从而阻止它们应用于不能折叠的蛋白质。在此，我们报道了人工裂解聚球藻DNAB解旋酶的纯化片段。PCC6803(SSP DNAB)内含子在自然条件下是活性的。蛋白质反式剪接反应的一级速率常数为7.1×10~(-4)S(~(-1))。酿酒酵母裂解空泡ATPase(SCE VMA)内含子是唯一一种在自然条件下有效的人工裂解内含子；然而，它需要辅助二聚结构域诱导内含子片段的复杂形成才能有效地进行蛋白质反式剪接。相反，二聚化结构域与分裂的SSP DNAB内含子片段的融合对活性没有影响。这种差异也反映在分裂的SSP DNAB内含子更高的热稳定性上。在优化的条件下对裂解的Sce VMA内含子的进一步研究表明，蛋白质反式剪接的一级速率常数为9.4x10(-4)S(-1)，涉及Cys1Ala突变体的C端切割的一级速率常数为1.7x10(-3)S(-1)。最后，我们证明了两个分裂的整数是正交的，这为从两个以上部分组装蛋白质提供了进一步的应用。
In protein trans-splicing, an intein domain split into two polypeptide chains mediates linkage of the flanking, amino acid sequences, the N- and C-terminal exteins, with a native peptide bond. This process can be exploited to assemble proteins from two separately prepared fragments, e.g., for the segmental labeling with isotopes for NMR studies or the incorporation of chemical and biophysical probes. Split inteins can be artificially generated by genetic means; however, the purified intein(N) and intein(C) fragments usually require a denaturation and renaturation treatment to fold into the active intein, thus preventing their application to proteins that cannot be refolded. Here, we report that the purified fragments of the artificially split DnaB helicase of Synechocystis spp. PCC6803 (Ssp DnaB) intein are active under native conditions. The first-order rate constant of the protein trans-splicing reaction was 7.1 X 10(-4) s(-1). The previously described split vacuolar ATPase of Saccharomyces cerevisiae (Sce VMA) intein is the only other artificially split intein that is active under native conditions; however, it requires induced complex formation of the intein fragments by auxiliary dimerization domains for efficient protein trans-splicing. In contrast, fusion of the dimerization domains to the split Ssp DnaB intein fragments had no effect on activity. This difference was also reflected by a higher thermostability of the split Ssp DnaB intein. Further investigations of the split Sce VMA intein under optimized conditions revealed a first-order rate constant of 9.4 x 10(-4) s(-1) for protein trans-splicing and 1.7 x 10(-3) s(-1) for C-terminal cleavage involving a Cys1Ala mutant. Finally, we show that the two split inteins are orthogonal, suggesting further applications for the assembly of proteins from more than two parts.