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Modulation of the catalytic activity of a metallonuclease by tagging with oligohistidine.
Journal of Inorganic Biochemistry ( IF 3.9 ) Pub Date : 2020-02-01 , DOI: 10.1016/j.jinorgbio.2020.111013
Heba A H Abd Elhameed 1 , Bálint Hajdu 1 , Attila Jancsó 1 , Albert Kéri 1 , Gábor Galbács 1 , Éva Hunyadi-Gulyás 2 , Béla Gyurcsik 1
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Peptide tags are extensively used for affinity purification of proteins. In an optimal case, these tags can be completely removed from the purified protein by a specific protease mediated hydrolysis. However, the interactions of these tags with the target protein may also be utilized for the modulation of the protein function. Here we show that the C-terminal hexahistidine (6 × His) tag can influence the catalytic activity of the nuclease domain of the Colicin E7 metallonuclease (NColE7) used by E. coli to kill competing bacteria under stress conditions. This enzyme non-specifically cleaves the DNA that results in cytotoxicity. We have successfully cloned the genes of NColE7 protein and its R447G mutant into a modified pET-21a DNA vector fusing the affinity tag to the protein upon expression, which would be otherwise not possible in the absence of the gene of the Im7 inhibitory protein. This reflects the inhibitory effect of the 6 × His fusion tag on the nuclease activity, which proved to be a complex process via both coordinative and non-specific steric interactions. The modulatory effect of Zn2+ ion was observed in the catalytic activity experiments. The DNA cleavage ability of the 6 × His tagged enzyme was first enhanced by an increase of metal ion concentration, while high excess of Zn2+ ions caused a lower rate of the DNA cleavage. Modelling of the coordinative effect of the fusion tag by external chelators suggested ternary complex formation instead of removal of the metal ion from the active center.

中文翻译:

通过标记寡聚组氨酸来调节金属核酸酶的催化​​活性。

肽标签广泛用于蛋白质的亲和纯化。在最佳情况下,可以通过特异性蛋白酶介导的水解从纯化的蛋白质中完全去除这些标签。但是,这些标签与靶蛋白的相互作用也可用于调节蛋白功能。在这里,我们显示C端六组氨酸(6×His)标签可以影响大肠杆菌在压力条件下杀死竞争性细菌的Colicin E7金属核酸酶(NColE7)的核酸酶结构域的催化活性。该酶非特异性地切割导致细胞毒性的DNA。我们已经成功地将NColE7蛋白的基因及其R447G突变体克隆到了经过修饰的pET-21a DNA载体中,该载体在表达后融合了对该蛋白的亲和标签,如果没有Im7抑制蛋白的基因,这是不可能的。这反映了6×His融合标签对核酸酶活性的抑制作用,这通过配位和非特异性空间相互作用被证明是一个复杂的过程。在催化活性实验中观察到了Zn2 +离子的调节作用。金属离子浓度的增加首先增强了6×His标记酶的DNA裂解能力,而大量过量的Zn2 +离子导致了较低的DNA裂解率。通过外部螯合剂对融合标签的协同作用进行建模,表明形成三元络合物而不是从活性中心去除金属离子。通过协调和非特定的空间相互作用,事实证明这是一个复杂的过程。在催化活性实验中观察到了Zn2 +离子的调节作用。金属离子浓度的增加首先增强了6×His标记酶的DNA裂解能力,而大量过量的Zn2 +离子导致了较低的DNA裂解率。通过外部螯合剂对融合标签的协同作用进行建模,表明形成三元络合物而不是从活性中心去除金属离子。通过协调和非特定的空间相互作用,事实证明这是一个复杂的过程。在催化活性实验中观察到了Zn2 +离子的调节作用。金属离子浓度的增加首先增强了6×His标记酶的DNA裂解能力,而大量过量的Zn2 +离子导致了较低的DNA裂解率。通过外部螯合剂对融合标签的协同作用进行建模,表明形成三元络合物而不是从活性中心去除金属离子。而大量过量的Zn2 +离子则导致DNA切割的速率降低。通过外部螯合剂对融合标签的协同作用进行建模,表明形成三元络合物而不是从活性中心去除金属离子。而大量过量的Zn2 +离子则导致DNA切割的速率降低。通过外部螯合剂对融合标签的协同作用进行建模,表明形成三元络合物而不是从活性中心去除金属离子。
更新日期:2020-02-03
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