Efficient production of recombinant T7 endonuclease I using silkworm-baculovirus expression vector system
Efficient production of recombinant T7 endonuclease I using silkworm-baculovirus expression vector system
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DOI：
10.1016/j.aspen.2020.05.001
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发表时间：
2020-08-01
影响因子：
1.5
通讯作者：
Lee, Jae Man
中科院分区：
文献类型：
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作者：
Kakino, Kohei;Masuda, Akitsu;Lee, Jae Man
最近，T7 核酸内切酶 I (T7E1) 裂解测定已被广泛用作检测 CRISPR/Cas9 靶向样本中突变的有效方法。该酶足以检测各种异源双链 DNA 样品中的单碱基和多碱基错配。然而，T7E1对于研究人员来说是相当昂贵的，仅将其用于筛选突变，尤其是在大量测试样本的情况下。关于这种酶的产生，据数据，仅报道了大肠杆菌系统，并且高度过表达的T7E1似乎对大肠杆菌宿主细胞有毒。因此，在本研究中，我们测试了家蚕杆状病毒表达载体系统（BEVS）是否适合生产重组T7核酸内切酶I（rT7E1）。带有N或C标签的rT7E1在培养的蚕细胞和蚕蛹中成功表达。我们的结果表明rT7E1-Ntag在蚕蛹中高表达，并且我们获得了高纯度的rT7E1蛋白。此外，来自蚕-BEVS的rT7E1充分识别并切割设计的和CRISPR/Cas9介导的DNA底物的错配，这与大肠杆菌系统的商业rT7E1相当。总而言之，我们的研究将通过提供一种经济有效且具有活性的 rT7E1 酶来极大地支持基因组编辑研究。
Recently, T7 Endonuclease I (T7E1) cleavage assay has been widely employed as an efficient approach for detecting mutations from CRISPR/Cas9 targeted samples. This enzyme is sufficient to detect single- and multiple-base mismatches from various heteroduplex DNA samples. However, T7E1 is quite expensive for researchers to use it only for screening mutations, especially in the condition of a large number of test samples. Regarding the production of this enzyme, to data, only the E. coli system has been reported and the highly overexpressed T7E1 seems toxic to the E. coli host cells. Thus, in this study, we tested whether the silkworm-baculovirus expression vector system (BEVS) is suitable to produce recombinant T7 Endonuclease I (rT7E1). The rT7E1 with N- or C-tags in cultured silkworm cells and silkworm pupae were successfully expressed. Our results demonstrated that the rT7E1-Ntag was highly expressed in silkworm pupae and we obtained rT7E1 proteins in high purity. Moreover, rT7E1 from silkworm-BEVS sufficiently recognized and cleaved the mismatches of designed and CRISPR/Cas9-mediated DNA substrates, which was equivalent to the commercial rT7E1 of the E. coli system. Taken together, our study would greatly support the genome-editing research by providing a cost-effective and active rT7E1 enzyme.