College of Life Sciences, Northwest Agriculture and Forestry University, Yangling, Shaanxi, 712100, China.
College of Horticulture, Northwest Agriculture and Forestry University, Yangling, Shaanxi, 712100, China.
Appl Biochem Biotechnol. 2018 Jul;185(3):641-654. doi: 10.1007/s12010-017-2672-4. Epub 2017 Dec 18.
Endonuclease I is a widely distributed periplasmic or extracellular enzyme. A method for the high-level production of recombinant AsEndI (endonuclease I from Aliivibrio salmonicida) in Escherichia coli with secretion expression is investigated. The coding sequence of AsEndI gene was assembled according to the E. coli codon usage bias, and AsEndI was expressed in the periplasm of E. coli TOP10 with a C-terminal 6× His-tagged fusion. The recombinant AsEndI (His-AsEndI) was purified by Ni-NTA resin with a yield of 1.29 × 10 U from 1-L LB medium. His-AsEndI could be classified into Ca/Mg-dependent nucleases and showed highest nuclease activity to dsDNA at pH 8.0 and 37 °C. His-AsEndI is highly active in a broad range of salt concentration range up to 1.0 M with optimal NaCl concentration at 0.4 M. His-AsEndI can effectively remove DNA contamination in RNA sample or in PCR reagents to the level that cannot be detected by highly sensitive nested PCR and without adverse effects on the subsequent PCR reaction. His-AsEndI can remove DNA contamination at high salt conditions, especially for the DNA that may be shielded by DNA-binding protein at low salt conditions.
内切酶 I 是一种广泛分布于细胞质周质或细胞外的酶。本研究探索了一种在大肠杆菌中通过分泌表达高水平生产重组 AsEndI（来自鲑鱼气单胞菌的内切酶 I）的方法。根据大肠杆菌密码子使用偏好性，组装了 AsEndI 基因的编码序列，并在大肠杆菌 TOP10 中带有 C 端 6×His 标记融合物在周质中表达。重组 AsEndI（His-AsEndI）通过 Ni-NTA 树脂纯化，从 1L LB 培养基中获得 1.29×10 U 的产量。His-AsEndI 可归类为 Ca/Mg 依赖性核酸酶，在 pH 8.0 和 37°C 时对 dsDNA 的核酸酶活性最高。His-AsEndI 在高达 1.0 M 的广泛盐浓度范围内具有很高的活性，最佳 NaCl 浓度为 0.4 M。His-AsEndI 可有效去除 RNA 样品或 PCR 试剂中的 DNA 污染，达到高度敏感的嵌套 PCR 无法检测的水平，且不会对后续 PCR 反应产生不利影响。His-AsEndI 可在高盐条件下去除 DNA 污染，特别是对于在低盐条件下可能被 DNA 结合蛋白屏蔽的 DNA。
