Arabidopsis ENDO2: Its Catalytic Role and Requirement of N-Glycosylation for Function
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- 作者：Chia-Yun Ko ; Yi-Ling Lai ; Wen-Yu Liu ; Chia-Hui Lin ; Yu-Ting Chen ; Long-Fang O. Chen ; Tsai-Yun Lin ; Jei-Fu Shaw
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2012
- 出版时间：May 23, 2012
- 年：2012
- 卷：60
- 期：20
- 页码：5169-5179
- 全文大小：622K
- 年卷期：v.60,no.20(May 23, 2012)
- ISSN：1520-5118
文摘
The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 脳 His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography鈥搈ass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.